JP2023503455A - Adeno-associated virus vector variants - Google Patents

Abstract

Targeting peptides and vectors containing sequences encoding said targeting peptides are provided herein, said targeting peptides delivering agents to specific substructures in the brain. Provided herein are viral vectors each comprising a modified capsid, wherein said modified capsid comprises at least one amino acid sequence that targets said viral vector to different brain structures. be. TIFF2023503455000034.tif49133

Description

REFERENCE TO RELATED APPLICATIONS This application supersedes U.S. Provisional Application No. 62/939,315, filed November 22, 2019, and U.S. Provisional Application No. 63/084,709, filed September 29, 2020 The entire contents of both applications are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING The present application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 19, 2020, is named CHOPP0038WO_ST25.txt and is 63.8 kilobytes in size.

1. Field The present invention relates generally to the fields of medicine, virology, and neurology. More specifically, the present invention relates to targeting peptides that target the delivery of viral vectors to different structures within the brain.

2. Description of Related Art Various strategies have been developed for generating AAV vector variants, including rational design and directed evolution. The rational design approach uses knowledge of the AAV capsid to make targeted changes to the capsid, such as tyrosine mutations on the capsid surface to increase transduction efficiency, to alter transduction efficiency or specificity. Directed evolution approaches do not require knowledge of the capsid structure and are performed by random mutagenesis, capsid shuffling, or random peptide insertion. These strategies generally use in vitro systems or mice, which are ideal for cell-based or mouse studies, but are not meant to translate to the clinic. In fact, AAV variants do not target different brain structures specifically or efficiently. Therefore, there is a need for AAV variants that can target different primate brain structures.

SUMMARY Provided herein are viral vectors each comprising a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to different brain structures.

In one embodiment, a modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide, wherein the targeting peptide allows the viral vector comprising the modified AAV capsid protein to be targeted to different organs or different brain structures,3 Modified AAV capsid proteins are provided that are ~10 amino acids long. In some aspects, the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

In some aspects, the modified AAV capsid protein is derived from the AAV1 capsid protein (see SEQ ID NO: 138) and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. In some aspects, the linker sequences are SSA N-terminal to the targeting peptide and AS C-terminal to the targeting peptide. In some aspects, the modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:141.

In some aspects, the modified AAV capsid protein is derived from the AAV2 capsid protein (see SEQ ID NO: 139) and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. In some aspects, the linker sequences are AAA N-terminal to the targeting peptide and AA C-terminal to the targeting peptide. In some aspects, the modified AAV2 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:142.

In some aspects, the modified AAV capsid protein is derived from the AAV9 capsid protein (see SEQ ID NO: 140) and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. In some aspects, the linker sequence is AAA N-terminal to the targeting peptide and AS C-terminal to the targeting peptide. In some aspects, the modified AAV9 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:143.

In some aspects, the target peptide comprises a sequence up to 10 amino acids long and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-137 and 144 in the sequence. In some aspects, the targeting peptide is 7 amino acids long.

In some aspects, the different brain structures are brain stem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidum, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. is. In certain aspects, the modified AAV capsid protein is a modified AAV1 capsid protein and the targeting peptides are selected from those listed in Table 1 to target the corresponding brain structures. In certain aspects, the modified AAV capsid protein is a modified AAV2 capsid protein and the targeting peptides are selected from those listed in Table 2 to target the corresponding brain structures. In certain aspects, the modified AAV capsid protein is a modified AAV9 capsid protein and the targeting peptides are selected from those listed in Table 3 to target the corresponding brain structures.

In some aspects the different organ is the brain, kidney, heart, liver, gonads, spleen, or liver. In certain aspects, the modified AAV capsid protein is a modified AAV1 capsid protein and the targeting peptides are selected from those listed in Table 4 to target the corresponding organ. In certain aspects, the modified AAV capsid protein is a modified AAV2 capsid protein and the targeting peptides are selected from those listed in Table 5 to target the corresponding organ. In certain aspects, the modified AAV capsid protein is a modified AAV9 capsid protein and the targeting peptides are selected from those listed in Table 6 to target the corresponding organ.

In one embodiment, provided herein is a nucleic acid comprising a sequence encoding a modified capsid protein of any one of the embodiments.

In one embodiment, provided herein is a recombinant adeno-associated virus (rAAV) comprising a modified capsid protein of any one of this embodiment. In some aspects, rAAV combinations are provided. For example, rAAV with modified AAV1 capsid protein and the targeting peptide of SEQ ID NO: 21, rAAV with modified AAV2 capsid protein and the targeting peptide of SEQ ID NO: 53, rAAV with modified AAV2 capsid protein and Targeting peptides of SEQ ID NO: 80 and combinations of rAAV and targeting peptides of SEQ ID NO: 113 with modified AAV9 capsid proteins are provided.

In one embodiment, provided herein is a viral vector comprising a nucleic acid encoding the modified capsid protein of any one of the embodiments. In some aspects, the viral vector further comprises a nucleic acid sequence encoding the nucleic acid of interest. In some aspects, the nucleic acid of interest is a therapeutic agent. In some aspects, the therapeutic agent is an enzyme or RNAi molecule.

In one embodiment, provided herein is a cell comprising the viral vector of any one of this embodiment. In some aspects, the cells are mammalian cells, such as human cells. In some aspects the cells are in vitro or in vivo.

In one embodiment, provided herein is a pharmaceutical composition comprising a viral vector of this embodiment and a pharmaceutically acceptable carrier.

In one embodiment, provided herein are methods for delivering agents to different brain structures of a subject comprising administering a virus of this embodiment to the subject. In some aspects, the different brain structures are brain stem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. is. In certain aspects, rAAV with a modified AAV1 capsid protein is used and targeting peptides are selected from those listed in Table 1 to target the corresponding brain structures. In certain aspects, rAAV with a modified AAV2 capsid protein is used and targeting peptides are selected from those listed in Table 2 to target the corresponding brain structures. In certain aspects, rAAV with a modified AAV9 capsid protein is used and targeting peptides are selected from those listed in Table 3 to target the corresponding brain structures. In various aspects, any combination of rAAVs is used. For example, rAAV with modified AAV1 capsid protein and the targeting peptide of SEQ ID NO: 21, rAAV with modified AAV2 capsid protein and the targeting peptide of SEQ ID NO: 53, rAAV with modified AAV2 capsid protein and The targeting peptide of SEQ ID NO:80 and a combination of rAAV and targeting peptide of SEQ ID NO:113 with a modified AAV9 capsid protein are used.

In one embodiment, provided herein are methods for delivering agents to different organs of a subject comprising administering a virus of this embodiment to the subject. In some aspects, the organ is the brain, kidney, heart, liver, gonads, spleen, or liver. In certain aspects, rAAV with a modified AAV1 capsid protein is used and targeting peptides are selected from those listed in Table 4 to target the corresponding organ. In certain aspects, rAAV with a modified AAV2 capsid protein is used and targeting peptides are selected from those listed in Table 5 to target the corresponding organ. In certain aspects, rAAV with a modified AAV9 capsid protein is used and targeting peptides are selected from those listed in Table 6 to target the corresponding organ. In various aspects, any combination of rAAVs is used.

In some aspects, the agent is an siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system. In some aspects, administration is to the central nervous system. In some aspects, administration is to the cisterna magna, intraventricular space, ependyma, ventricle, subarachnoid space, and/or intrathecal space. In some aspects, the ventricle is a rostral ventricle, and/or a caudal ventricle, and/or a right ventricle, and/or a left ventricle, and/or a right rostral ventricle, and /or the left rostral ventricle, and/or the right caudal ventricle, and/or the left caudal ventricle.

In some aspects, multiple virus particles are administered. In some aspects, the virus is administered at a dose of about 1×10 ⁶ to about 1×10 ¹⁸ vector genomes per kilogram (vg/kg). In some aspects, the virus is about 1×10 ⁷ to 1×10 ¹⁷ , about 1×10 ⁸ to 1×10 ¹⁶ , about 1×10 ⁹ to 1×10 ¹⁵ , about 1×10 vg per kg of patient. ¹⁰ to 1×10 ¹⁴ , about 1×10 ¹⁰ to 1×10 ¹³ , about 1×10 ¹⁰ to 1×10 ¹³ , about 1×10 ¹⁰ to 1×10 ¹¹ , about 1×10 ¹¹ to 1×10 ¹² , about 1×10 ¹² to 1×10 ¹³ , or about 1×10 ¹³ to 1×10 ¹⁴ . In some aspects, the subject is human.

In one embodiment, provided herein is a method of treating a disease in a mammal comprising administering a virus of this embodiment to the mammal. In some aspects the disease is a neurodegenerative disease. In some aspects, the neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, Alzheimer's disease, polyglutamine repeat disease, or Parkinson's disease. be. In some aspects, the mammal is human.

As used herein, "essentially free" with respect to a specified ingredient means that none of the specified ingredient is intentionally formulated into the composition and/or as a contaminant or in trace amounts. is meant to exist only in The total amount of specific ingredients resulting from unintentional contamination of the composition is therefore considerably lower than 0.05%, preferably lower than 0.01%. Most preferred are compositions in which the amount of such specific ingredients cannot be detected by standard analytical methods.

As used herein, "a" or "an" may mean one or more. As used herein in the claims, the word "a" or "an" when used with the word "comprising" refers to one or two It can mean the above.

The use of the term "or" in the claims is used to mean "and/or" unless expressly indicated to mean only alternatives or mutually exclusive alternatives, but the present disclosure supports the definition of alternatives only and "and/or". As used herein, "another" may mean at least a second or more.

Throughout this application, the term "about" means that a value is within 10% of a stated value, the inherent error variation of the equipment or methods used to determine that value, the variation that exists between study subjects, or Used to indicate that it contains a value.

Other objects, features, and advantages of the present invention will become apparent from the detailed description below. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

The following drawings form part of this specification and are provided to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.
Schematic of the AAV peptide display library. Schematic representation of the in vivo screening strategy. Graphical representation of input library diversity. Input viral library diversity measured from aliquots of AAV1, AAV2, and AAV9 viral vectors before round 1 ICV injection. Graphical representation of round-over-round barcode enrichment. Total number of unique barcodes recovered after round 1 and round 2 enrichment in Rhesus macaque for each tissue collected. Round 2 values for DNA and RNA are shown. See description for Figure 4-1. See description for Figure 4-1. Illustration of round-over-round enrichment of barcodes in AAV1 serotypes in the cerebellar cortex. Illustration of round-over-round enrichment of barcodes in AAV2 serotypes in the cerebellar cortex. Illustration of round-over-round enrichment of barcodes in AAV9 serotypes in the cerebellar cortex. Illustration of enrichment of AAV9 1999. Heatmap depiction of barcode enrichment from AAV9, with cells color-coded by the percentage of barcodes detected from the indicated tissues. Barcodes recovered from DNA are shown on the left and barcodes recovered from RNA are shown on the right. See description for Figure 6-1. Heatmap depiction of opool barcode enrichment from AAV1. Heatmap depiction of opool barcode enrichment from AAV2. Heatmap depiction of opool barcode enrichment from AAV9. AAV9 1999 in vivo rhesus validation. The eGFP expression construct was packaged into AAV9 1999 driven by the CAG promoter. AAV9 1999 1.5E13 vg was delivered to 5 year old female rhesus monkeys by ICV injection. Representative images of H&E-stained cerebellum depicting the transduction pattern of AAV9 1999 are shown. Figures 9A-D. AAV9 1999 in vivo mouse validation. The eGFP expression construct was packaged into AAV9 1999 driven by the CAG promoter. AAV9 1999 and AAV9 capsids containing eGFP constructs were delivered to C57BL/6 p0 mouse pups by ICV injection at 1E10 vg. Representative images of eGFP fluorescence signal are whole brain (Figure 9A), whole brain sagittal section (Figure 9B), S1 cortical section (Figure 9C, left), hippocampal section (Figure 9C, middle), cerebellar sagittal section. (Fig. 9C, right), and a lumbar spinal coronal section (Fig. 9D). Fluorescence image of the in vivo rhesus monkey lateral ventricle of the AAV mixture. Fluorescence image of the in vivo Rhesus fourth ventricle of the AAV mixture. Fluorescent images of in vivo rhesus meninges of the AAV mixture. Fluorescent images of cochlear turns after cochlear administration of AAV9 1999 capsids containing eGFP constructs to mice. Fluorescent images of inner hair cells after cochlear administration of AAV9 1999 capsids containing eGFP constructs to mice. Fluorescent images of the organ of Corti and distal cochlear axis after cochlear administration of AAV9 1999 capsids containing eGFP constructs to mice.

DETAILED DESCRIPTION Provided herein are viral vectors each comprising a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to different brain structures. be. In certain embodiments, the brain structure is in the brain stem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. be. Targeting peptides for each brain structure are provided in Tables 1-3.

In certain embodiments, the viral vector is an adeno-associated viral vector (AAV). In certain embodiments, the AAV is AAV1, AAV2, or AAV9. An exemplary wild-type reference AAV1 capsid protein sequence is provided in SEQ ID NO:138. An exemplary wild-type reference AAV2 capsid protein sequence is provided in SEQ ID NO:139. An exemplary wild-type reference AAV9 capsid protein sequence is provided in SEQ ID NO:140. In certain aspects, the targeting peptide is inserted at position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, or position 588 of the AAV9 capsid. An exemplary modified AAV1 capsid protein sequence is provided in SEQ ID NO: 141, which shows a targeting peptide insertion after position 590 as SSAX ₇ AS, where the leading SSA and trailing AS are linker sequences. and _X7 represents the targeting peptide. An exemplary modified AAV2 capsid protein sequence is provided in SEQ ID NO: 142, which shows a targeting peptide insertion after position 587 as AAAX ₇ AA, where the leading AAA and trailing AA are linker sequences. and _X7 represents the targeting peptide. An exemplary modified AAV9 capsid protein sequence is provided in SEQ ID NO: 143, which shows the targeting peptide insertion after position 588 as AAAX ₇ AS, where the leading AAA and trailing AS are linker sequences. and _X7 represents the targeting peptide.

(Table 1) AAV1 targeting peptides for each brain structure

(Table 2) AAV2 targeting peptides for each brain structure

(Table 3) AAV9 targeting peptides for each brain structure

(Table 4) AAV1 targeting peptides to various organs

(Table 5) AAV2 targeting peptides to various organs

(Table 6) AAV9 targeting peptides to various organs

I. Adeno-Associated Virus (AAV) Vectors Adeno-associated virus (AAV) is a small, non-pathogenic virus of the Parvoviridae family. To date, a number of serologically distinct AAVs have been identified, with more than a dozen also from humans or primates. AAV differs from other members of this family in that it relies on a helper virus for replication.

The AAV genome exists in an extrachromosomal state without integration into the genome of the host cell, has a broad host range, transduces both dividing and non-dividing cells in vitro and in vivo, and transduces can maintain high levels of expression of the engineered genes. AAV virions are thermostable, resistant to changes in solvents, detergents, pH, and temperature, and can be column purified and/or concentrated by CsCl gradients or other means. The AAV genome comprises single-stranded deoxyribonucleic acid (ssDNA), either positive or negative. The approximately 4.7 kb genome of AAV consists of a segment of single-stranded DNA of either positive or negative polarity. Flanking the genome are short inverted terminal repeats (ITRs) that can fold into a hairpin structure and serve as origins of viral DNA replication.

AAV "genome" refers to the recombinant nucleic acid sequences that are ultimately packaged or encapsulated to form AAV particles. AAV particles often contain the AAV genome packaged with AAV capsid proteins. When a recombinant plasmid is used to construct or produce a recombinant vector, the AAV vector genome does not include portions of the "plasmid" that do not correspond to the vector genome sequences of the recombinant plasmid. This non-vector genome portion of a recombinant plasmid is referred to as the "plasmid backbone" and is important for plasmid cloning and amplification, processes necessary for plasmid propagation and production, but which itself is packaged into viral particles. not wrapped or encapsulated. An AAV vector "genome" thus refers to the nucleic acid that is packaged or enclosed by the AAV capsid proteins.

AAV virions (particles) are non-enveloped icosahedral particles approximately 25 nm in diameter that contain the AAV capsid. AAV particles contain an icosahedral symmetry composed of three related capsid proteins VP1, VP2, and VP3 that interact with each other to form a capsid. The genome of most native AAVs often contains two open reading frames (ORFs), sometimes referred to as the left ORF and the right ORF. The right ORF often encodes the capsid proteins VP1, VP2 and VP3. These proteins are often found in a 1:1:10 ratio, respectively, but the ratios can vary, and both are derived from the right-hand ORF. The VP1, VP2, and VP3 capsid proteins differ from each other by alternative splicing and unusual initiation codon usage. Deletion analysis has shown that removal or remodeling of VP1 translated from alternatively spliced messages results in reduced yields of infectious particles. Mutations within the VP3 coding region result in defective production of single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of the AAV particle encodes one, two or all three of the VP1, VP2 and VP3 polypeptides.

The left-hand ORF encodes the nonstructural Rep proteins Rep40, Rep52, Rep68, and Rep78, which are often involved in the regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Two of the Rep proteins have been associated with preferential inclusion of the AAV genome into a region of the human chromosome 19 q arm. Rep68/78 has been shown to have NTP binding activity in addition to DNA and RNA helicase activities. Some Rep proteins have nuclear localization signals with several potential phosphorylation sites. In certain embodiments, the AAV (eg, rAAV) genome encodes part or all of the Rep protein. In certain embodiments, the AAV (eg, rAAV) genome does not encode a Rep protein. In certain embodiments, one or more of the Rep proteins can be delivered in trans and thus are not included in the AAV particle comprising the nucleic acid encoding the polypeptide.

The ends of the AAV genome contain short inverted terminal repeats (ITRs) that have the potential to fold into T-shaped hairpin structures that serve as origins of viral DNA replication. The AAV genome thus comprises one or more (eg, a pair) of ITR sequences that flank the single-stranded viral DNA genome. ITR sequences often have a length of about 145 bases each. Within the ITR region, two elements that are thought to be central to ITR function have been described: the GAGC repeat motif and the terminal resolution site (trs). Repeat motifs have been shown to bind Rep when the ITR is in either the linear or hairpin conformation. This binding is thought to position Rep68/78 for cleavage at trs to occur in a site- and strand-specific manner. These two elements appear to be central to viral containment, in addition to their role in replication. The chromosome 19 integration locus contains a Rep binding site flanked by trs. These elements have been shown to be functional and required for locus-specific inclusion.

The term "recombinant" as a modifier of vectors, such as recombinant viral vectors, e.g., recombinant lentiviral or recombinant parvoviral (e.g., AAV) vectors, and sequences such as recombinant nucleic acid sequences or recombinant polypeptides. The term "recombinant" as a modifier means that the composition has been manipulated (ie, engineered) in a manner not commonly occurring in nature. Particular examples of recombinant vectors, such as AAV vectors, retroviral vectors or lentiviral vectors, are those in which a nucleic acid sequence not normally present in the wild-type viral genome has been inserted into the viral genome. An example of a recombinant nucleic acid sequence is one in which a nucleic acid (e.g., a gene) is present in a vector with or without the 5', 3', and/or intron regions with which the gene is normally associated in the viral genome. It encodes an inhibitory RNA cloned into . The term "recombinant" is not always used herein in reference to vectors such as viral vectors and sequences such as polynucleotides, including nucleic acid sequences, polynucleotides, transgenes, etc. is expressly included notwithstanding such omission.

A recombinant viral "vector" is derived from the wild-type genome of a virus by using molecular methods to remove portions of the wild-type genome from the virus and replace them with non-native nucleic acids, such as nucleic acid sequences. Typically, for AAV for example, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A "recombinant" viral vector (e.g., rAAV) is one in which a portion of the viral genome, compared to the viral genomic nucleic acid, is a nucleic acid encoding a transactivator or a nucleic acid encoding an inhibitory RNA or a nucleic acid encoding a therapeutic protein. It is distinguished from the viral (eg AAV) genome in that it has been replaced with non-native sequences such as. Therefore, the incorporation of such non-native nucleic acid sequences defines the viral vector as a "recombinant" vector, which in the case of AAV may be referred to as a "rAAV vector".

In certain embodiments, the AAV (eg rAAV) comprises two ITRs. In certain embodiments, the AAV (eg, rAAV) comprises a pair of ITRs. In certain embodiments, an AAV (eg, rAAV) has at least a pair of ITRs that flank the nucleic acid sequence encoding a polypeptide with function or activity (ie, are present at the 5' and 3' ends of the nucleic acid sequence, respectively). include.

AAV vectors (eg, rAAV vectors) can be packaged and are referred to herein as "AAV particles" for infection (transduction) of cells ex vivo, in vitro or in vivo. When a recombinant AAV vector is enclosed or packaged in an AAV particle, the particle can also be referred to as a "rAAV particle." In certain embodiments, the AAV particles are rAAV particles. A rAAV particle often comprises a rAAV vector or portion thereof. The rAAV particle can be one or more rAAV particles (eg, multiple AAV particles). The rAAV particles typically contain proteins (eg, capsid proteins) that enclose or package the rAAV vector genome. Note that references to rAAV vectors can also be used to refer to rAAV particles.

Any suitable AAV particles (eg, rAAV particles) can be used in the methods or uses herein. The rAAV particles and/or the genome contained therein can be derived from any suitable serotype or strain of AAV. The rAAV particles and/or the genomes contained therein can be derived from two or more serotypes or strains of AAV. The rAAV can thus comprise proteins and/or nucleic acids or portions thereof of any serotype or strain of AAV, the AAV particles being suitable for infection and/or transduction of mammalian cells. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 and AAV-2i8. .

In certain embodiments, the plurality of rAAV particles comprises particles of the same strain or serotype (or subgroup or variant) or particles derived from the same strain or serotype (or subgroup or variant). In certain embodiments, the plurality of rAAV particles comprises a mixture of two or more different (eg, different serotypes and/or different strains) rAAV particles.

As used herein, the term "serotype" is a characteristic used to refer to AAV having a capsid that is serologically distinct from other AAV serotypes. Serological signature is determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. Such cross-reactivity differences are usually due to capsid protein sequence/antigenic determinant differences (eg due to differences in the VP1, VP2 and/or VP3 sequences of AAV serotypes). Even though AAV variants, including capsid variants, may not be serologically distinct from reference AAV serotypes or other AAV serotypes, they may have at least one They differ in nucleotide or amino acid residues.

In certain embodiments, rAAV vectors based on the first serotype genome correspond to one or more serotypes of the capsid proteins that package the vector. For example, the serotypes of one or more of the AAV nucleic acids (eg, ITRs) that make up the AAV vector genome correspond to the serotypes of the capsids that make up the rAAV particle.

In certain embodiments, rAAV vector genomes can be based on AAV (eg, AAV2) serotype genomes derived from one or more serotypes of the vector-packaging AAV capsid proteins. For example, the rAAV vector genome can comprise nucleic acid (e.g., ITR) from AAV2, while at least one or more of the three capsid proteins are of different serotypes, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, Derived from AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 serotypes or variants thereof.

In certain embodiments, the rAAV particle or its vector genome associated with the reference serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV- Polynucleotides, polypeptides or subsequences of 2i8 particles and at least 60% or more (e.g. 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) have a polynucleotide, polypeptide or subsequence thereof comprising or consisting of a sequence that is identical. In particular embodiments, the rAAV particle or its vector genome associated with the reference serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 At least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, etc.) have capsid or ITR sequences that contain or consist of sequences that are identical.

In certain embodiments, the methods herein involve the use, administration , or including delivery.

In certain embodiments, the methods herein involve the use, administration or delivery of rAAV2 particles. In certain embodiments, the rAAV2 particle comprises an AAV2 capsid. In certain embodiments, the rAAV2 particles are at least 60%, 65%, 70%, 75%, or more identical, e.g., 80%, 85%, 85%, to the corresponding capsid proteins of native AAV2 particles or wild-type AAV2 particles. %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, Contains one or more capsid proteins (eg VP1, VP2 and/or VP3) that are 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV2 particles are at least 75% or more identical, e.g., 80%, 85%, 86%, 87%, 88%, 89%, to the corresponding capsid proteins of native AAV2 particles or wild-type AAV2 particles. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc. up to 100% It contains identical VP1, VP2 and VP3 capsid proteins. In certain embodiments, the rAAV2 particles are native AAV2 particles or variants of wild-type AAV2 particles. In some aspects, one or more capsid proteins of the AAV2 variant are 1, 2, 3, 4, 5, 5-10, 10-15 compared to the capsid protein of native AAV2 particles or wild-type AAV2 particles. , with 15-20 or more amino acid substitutions.

In certain embodiments, the rAAV9 particle comprises an AAV9 capsid. In certain embodiments, the rAAV9 particles are at least 60%, 65%, 70%, 75%, or more identical, e.g., 80%, 85%, 85%, to the corresponding capsid proteins of native AAV9 particles or wild-type AAV9 particles. %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, Contains one or more capsid proteins (eg VP1, VP2 and/or VP3) that are 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV9 particles are at least 75% or more identical, e.g., 80%, 85%, 86%, 87%, 88%, 89%, to the corresponding capsid proteins of native AAV9 particles or wild-type AAV9 particles. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc. up to 100% It contains identical VP1, VP2 and VP3 capsid proteins. In certain embodiments, the rAAV9 particles are native AAV9 particles or variants of wild-type AAV9 particles. In some aspects, one or more capsid proteins of the AAV9 variant are 1, 2, 3, 4, 5, 5-10, 10-15 compared to the capsid protein of native AAV9 particles or wild-type AAV9 particles. , with 15-20 or more amino acid substitutions.

In certain embodiments, the rAAV particles have one or more desired ITR functions (e.g., the ability to form hairpins that allow DNA replication, inclusion of AAV DNA into the host cell genome, and/or native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 or at least 75% or more identical to the corresponding ITR of AAV-2i8, e.g. One or two ITRs (e.g., a pair of ITRs) that are up to 100% identical, such as 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5% including.

In certain embodiments, the rAAV2 particles exhibit one or more of the desired ITR functions (e.g., the ability to form hairpins that allow DNA replication, inclusion of AAV DNA into the host cell genome, and/or at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89, to the corresponding ITR of native or wild-type AAV2 particles as long as they retain the %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc. up to 100 Contains one or two ITRs (eg, a pair of ITRs) that are %identical.

In certain embodiments, the rAAV9 particles have one or more desired ITR functions (e.g., the ability to form hairpins that allow DNA replication, inclusion of AAV DNA into the host cell genome, and/or at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89, to the corresponding ITR of native or wild-type AAV2 particles as long as they retain the %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc. up to 100 Contains one or two ITRs (eg, a pair of ITRs) that are %identical.

The rAAV particles can contain ITRs with any suitable number of "GAGC" repeats. In certain embodiments, the ITRs of the AAV2 particles comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In certain embodiments, the rAAV2 particles comprise ITRs containing three "GAGC" repeats. In certain embodiments, the rAAV2 particles comprise ITRs with less than 4 "GAGC" repeats. In certain embodiments, the rAAV2 particles comprise ITRs with more than 4 "GAGC" repeats. In certain embodiments, the ITRs of rAAV2 particles contain a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is a C instead of a T.

An example of a suitable length of DNA that can be incorporated into a rAAV vector for packaging/encapsidation into rAAV particles can be about 5 kilobases (kb) or less. In certain embodiments, the length of DNA is less than about 5 kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb.

rAAV vectors containing nucleic acid sequences that direct expression of RNAi or polypeptides can be generated using any suitable recombinant technique known in the art (see, eg, Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transducible AAV particles and propagated using AAV viral packaging systems. Transducible AAV particles have the ability to bind and enter mammalian cells and subsequently deliver their nucleic acid cargo (eg, heterologous genes) to the cell's nucleus. Thus, transducible, intact rAAV particles are configured to transduce mammalian cells. rAAV particles designed to transduce mammalian cells are often replication incompetent and require additional protein machinery for self-renewal. Thus, rAAV particles configured to transduce mammalian cells have been engineered to bind to and enter mammalian cells and deliver nucleic acids to the cells, wherein the delivered nucleic acids are , often located between a pair of AAV ITRs in the rAAV genome.

Suitable host cells for producing transducible AAV particles include microorganisms, yeast cells, which can be or have been used as recipients of heterologous rAAV vectors. , insect cells and mammalian cells. The stable human cell line HEK293 (readily available, eg, from the American Type Culture Collection under accession number ATCC CRL1573) can be used. In certain embodiments, a modified human embryonic kidney cell line (e.g., HEK293) that has been transformed with an adenovirus type 5 DNA fragment and expresses the adenoviral E1a and E1b genes is used to generate recombinant AAV particles. used. The modified HEK293 cell line is easily transfected, making it a particularly convenient platform for producing rAAV particles. Methods for producing high titer AAV particles capable of transducing mammalian cells are known in the art. For example, AAV particles can be made as described in Wright, 2008 and Wright, 2009.

In certain embodiments, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct prior to transfection of the AAV expression vector or concurrently with transfection of the AAV expression vector. . Thus, in some cases, AAV helper constructs are used to at least transiently express AAV rep and/or cap genes in order to complement missing AAV functions required for productive AAV transduction. used. AAV helper constructs often lack AAV ITRs and are unable to replicate or package themselves. These constructs can take the form of plasmids, phages, transposons, cosmids, viruses, or virions. Several AAV helper constructs have been described, including the popular plasmids pAAV/Ad and pIM29+45, which encode both Rep and Cap expression products. A number of other vectors encoding Rep and/or Cap expression products are also known.

An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence together with the necessary regulatory regions necessary for expression in a host cell. Expression vectors can contain at least an origin of replication for propagation in a cell and optionally additional elements such as heterologous nucleic acid sequences, expression control elements (eg promoters, enhancers), introns, ITRs, and polyadenylation signals.

II. Therapeutic Agents In some embodiments, viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Using such methods, inhibitory RNAs, non-coding RNAs, and/or nucleic acids encoding therapeutic proteins can be administered to cells in culture or in a host organism.

A. Inhibitory RNA
"RNA interference (RNAi)" is the process of sequence-specific post-transcriptional gene silencing initiated by siRNA. During RNAi, siRNAs induce degradation of target mRNAs, resulting in sequence-specific inhibition of gene expression.

"Inhibitory RNA", "RNAi", "small interfering RNA" or "short interfering RNA" or "siRNA" molecules, "short hairpin RNA" or "shRNA" molecules, or "miRNA" are An RNA duplex of nucleotides that targets a nucleic acid sequence. As used herein, the term "siRNA" is a generic term that encompasses a subset of shRNAs and miRNAs. "RNA duplex" refers to the structure formed by complementary pairing between two regions of an RNA molecule. An siRNA "targets" a gene because the nucleotide sequence of the double-stranded portion of the siRNA is complementary to that of the gene. In certain embodiments, the siRNA targets a sequence encoding huntingtin. In some embodiments, the siRNA duplex length is less than 30 base pairs. In some embodiments, the duplex is 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or It can be 10 base pairs long. In some embodiments, the length of the duplex is 19-25 base pairs long. In certain embodiments, the length of the duplex is 19 base pairs long or 21 base pairs long. The RNA duplex portion of the siRNA can be part of a hairpin structure. Hairpin structures contain, in addition to the duplex portion, a loop portion located between the two sequences forming the duplex. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 Nucleotide length. In certain embodiments, the loop is 18 nucleotides long. Hairpin structures can also contain 3' and/or 5' overhangs. In some embodiments, the overhangs are 3' and/or 5' overhangs of 0, 1, 2, 3, 4, or 5 nucleotides in length.

shRNAs are composed of stem-loop structures designed to contain a 5' flanking region, an siRNA region segment, a loop region, a 3' siRNA region and a 3' flanking region. Most RNAi expression strategies have used short hairpin RNAs (shRNAs) driven by strong polIII-based promoters. Although many shRNAs exhibit effective knockdown of target sequences in vitro and in vivo, some shRNAs exhibiting effective knockdown of target genes were also found to be toxic in vivo.

miRNAs are small cellular RNAs (approximately 22 nt) that are processed from precursor stem-loop transcripts. Known miRNA stem-loops can be modified to contain RNAi sequences specific for the gene of interest. miRNA molecules may be preferred over shRNA molecules. This is because miRNAs are endogenously expressed. Therefore, miRNA molecules have been shown to be less likely to induce the dsRNA-responsive interferon pathway, processed more efficiently than shRNA, and silencing 80% more effectively than shRNA.

A recently discovered alternative approach is the use of artificial miRNAs (pri-miRNA scaffolds that shuttle siRNA sequences) as RNAi vectors. Artificial miRNAs more naturally resemble endogenous RNAi substrates, allowing Pol-II transcription (e.g. enabling tissue-specific expression of RNAi) and polycistronic strategies (e.g. delivering multiple siRNA sequences). ) are also more suitable. See US Pat. No. 10,093,927, incorporated herein by reference.

The "shRNA" transcription unit consists of a sense and antisense sequence joined by a loop of unpaired nucleotides. shRNA is exported from the nucleus by exportin-5, and when it enters the cytoplasm, it undergoes processing by Dicer to generate functional siRNA. The stem-loop of a "miRNA" consists of sense and antisense sequences joined by a loop of unpaired nucleotides, which are typically expressed as part of a larger primary transcript (pri-miRNA). is excised by the Drosha-DGCR8 complex to produce an intermediate known as pre-miRNA, which is then exported from the nucleus by exportin-5 and enters the cytoplasm where it is processed by Dicer to Generate functional siRNA. As used interchangeably herein, "artificial miRNA" or "artificial miRNA shuttle vector" refers to a region (at least about 9-20 nucleotides) of the double-stranded stem loop that is excised by Drosha and Dicer processing A primary miRNA transcript that has been replaced with an siRNA sequence for a target gene while retaining the structural elements within the stem-loop required for efficient Drosha processing. The term "artificial" derives from the fact that the flanking sequences (approximately 35 nucleotides upstream and approximately 40 nucleotides downstream) are derived from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein, the term "miRNA" encompasses both naturally occurring miRNA sequences and artificially produced miRNA shuttle vectors.

An siRNA can be encoded by a nucleic acid sequence, which can also include a promoter. This nucleic acid sequence can also contain a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six T's.

There are several factors that need to be considered in RNAi design, including the nature of the siRNA, the persistence of the silencing effect and the choice of delivery system. In order to produce an RNAi effect, siRNAs introduced into an organism typically contain exonic sequences. Furthermore, since the RNAi process is homology-dependent, the sequences should be sequenced to maximize gene specificity while minimizing the potential for cross-interference between homologous but not gene-specific sequences. must be chosen carefully. Preferably, the siRNA exhibits greater than 80%, 85%, 90%, 95%, or 98% identity, or even 100% identity, between the sequence of the siRNA and the gene to be inhibited. Sequences with less than about 80% identity to the target gene are significantly less efficient. Therefore, the higher the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

Additionally, the size of the siRNA is also an important consideration. In some embodiments, the invention relates to siRNA molecules comprising at least about 19-25 nucleotides and capable of modulating gene expression. For the present invention, siRNAs are preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably, the siRNA is about 19 nucleotides to about 25 nucleotides in length.

An siRNA target generally refers to a polynucleotide that contains a region that encodes a polypeptide, or a polynucleotide region that regulates replication, transcription or translation or other processes important for the expression of a polypeptide, or a polypeptide. A polynucleotide that includes both a coding region and a region operably linked thereto that regulates expression. Any gene that is expressed in the cell can be targeted. Preferably, the target gene is involved in or associated with a cellular activity progression that is important to disease or of particular interest to study.

B. Noncoding RNA
More than 90% of the human genome is transcribed but does not encode proteins, as evidenced by cDNA cloning projects and genome tiling arrays. These transcripts are called non-protein-coding RNAs (ncRNAs). Various ncRNA transcripts, such as ribosomal RNA, transfer RNA, competitive endogenous RNA (ceRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA), are essential for cellular function. Similarly, many short ncRNAs such as microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs) are also important in eukaryotic cells. known to play a regulatory role. Recent studies have demonstrated a group of long ncRNA (lncRNA) transcripts that exhibit cell type-specific expression and localize to specific subcellular compartments. lncRNAs are also known to play important roles during cell development and differentiation, supporting the view that they were selected during evolution.

LncRNAs appear to have many different functions. In many cases, they appear to play a role in regulating protein activity or localization, or serve as an organizational framework for intracellular structures. In other cases, lncRNAs may be processed to produce multiple small RNAs, or other RNA processing methods may be adjusted. The latest version of data generated by the public research consortium GenCode (version number 27) cataloged just under 16,000 lncRNAs in the human genome, generating nearly 28,000 transcripts; other databases included. More than 40,000 lncRNAs are known, if any.

Interestingly, lncRNAs influence the expression of specific target proteins at specific genomic loci, modulate the activity of protein binding partners, direct chromatin modification complexes to their sites of action, and are post-transcriptionally processed. A large number of 5' capped small RNAs can be generated. Epigenetic pathways can also regulate the differential expression of lncRNAs.

A growing body of evidence also suggests that aberrantly expressed lncRNAs play important roles in normal physiological processes and in multiple disease states. lncRNAs are misregulated in a variety of diseases, including ischemia, heart disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. This misregulation has also been shown in various types of cancer such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma and leukemia. Several lncRNAs, such as gadd74 and lncRNA-RoR5, regulate cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors and p53, thus providing additional flexibility and robustness to cell cycle progression. I will provide a. In addition, some lncRNAs are involved in mitotic processes, such as centromere satellite RNAs, which are essential for centromere formation and thus important for chromosome segregation during mitosis in humans and flies. Another nuclear lncRNA, MA-lincl, regulates M-phase exit by functioning in cis to repress the expression of the neighboring gene Pura, a regulator of cell proliferation.

lncRNAs are a group generally defined as transcripts of more than 200 nucleotides (eg, about 200 to about 1200 nt, about 2500 nt, or more) that lack an extended open reading frame (ORF). The term "non-coding RNA" (ncRNA) includes lncRNAs and short transcripts of less than about 200 nt, eg, about 30-200 nt.

Thus, in some embodiments, delivery of ncRNAs, eg, to specific brain structures of interest, corrects aberrant RNA expression levels or modulates levels of disease-causing lncRNAs. Accordingly, in some embodiments, the present invention provides rAAVs whose viral genomes have been engineered to encode therapeutic non-coding RNAs (ncRNAs). In some embodiments, the ncRNA is a long noncoding RNA (lncRNA) of about 200 nucleotides (nt) or more in length. In some embodiments, the therapeutic agent is a ncRNA from about 25 nt or from about 30 nt to about 200 nt in length. In some embodiments, lncRNAs are from about 200 nt to about 1,200 nt in length. In some embodiments, lncRNAs are from about 200 nt to about 1,100, about 1,000, about 900, about 800, about 700, about 600, about 500, about 400, or about 300 nt in length.

C. CRISPR System Gene editing is a technology that allows targeted gene modification in living cells. In recent years, the implementation of on-demand gene editing using the bacterial CRISPR immune system has revolutionized the way scientists approach genome editing. The Cas9 protein of the CRISPR system, an RNA-guided DNA endonuclease, can be engineered to target new sites relatively easily by changing its guide RNA sequence. This discovery made sequence-specific gene editing functionally effective.

Generally, the "CRISPR system" includes transcripts and other elements involved in directing the expression of CRISPR-associated ("Cas") genes or their activity, e.g., sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g. tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats that have undergone tracrRNA processing in the case of endogenous CRISPR systems), guide sequences (in the case of endogenous CRISPR systems Also referred to as "spacers") and/or other sequences and transcripts from the CRISPR locus are collectively referred to.

A CRISPR/Cas nuclease or CRISPR/Cas nuclease system comprises a non-coding RNA molecule (guide) RNA that binds sequence-specifically to DNA and a Cas protein (e.g. Cas9) with nuclease functionality (e.g. two nuclease domains). can contain. One or more elements of the CRISPR system can be derived from a type I, type II or type III CRISPR system, for example from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes. be able to.

CRISPR systems are capable of inducing a double stranded break (DSB) at a target site followed by a split, as discussed herein. In another embodiment, a Cas9 variant, considered a "nickase", is used to nick a single strand at the target site. A pair of nickases, each directed by a pair of different gRNAs targeting sequences in such a way that a 5' overhang is introduced when the nicks are co-introduced, e.g. to improve specificity. can be used. In another embodiment, catalytically inactive Cas9 is fused to heterologous effector domains, such as transcriptional repressors (eg, KRAB) or transcriptional activators, to affect gene expression. Alternatively, the catalytically inactive Cas9 based CRISPR system further comprises a transcriptional repressor or transcriptional activator fused to a ribosome binding protein.

In some aspects, a Cas nuclease and a gRNA (including a fusion of a target sequence-specific crRNA and an invariant tracrRNA) are introduced into the cell. Generally, the target site at the 5' end of the gRNA uses complementary base pairing to target the Cas nuclease to the target site, eg, the gene. A target site may be selected based on its position, typically immediately 5' of a protospacer adjacent motif (PAM) sequence, eg, NGG or NAG. In this regard, gRNAs can be produced by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. target the sequence of CRISPR systems are generally characterized by elements that promote formation of the CRISPR complex at the site of the target sequence. Generally, a "target sequence" generally refers to a sequence to which a guide sequence is designed to be complementary, and hybridization between the target sequence and the guide sequence facilitates formation of the CRISPR complex. . Perfect complementarity is not necessary provided that there is sufficient complementarity to cause hybridization and promote formation of the CRISPR complex.

A target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence can be located within the nucleus or cytoplasm of the cell, such as within an organelle of the cell. Generally, a sequence or template that can be used for recombination into a targeted locus containing a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence." In some aspects, an exogenous template polynucleotide can be referred to as an editing template. In some aspects the recombination is homologous recombination.

Typically, for an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence to form a complex with one or more Cas proteins) occurs within the target sequence or within the target sequence. Cleavage of one or both strands in the vicinity of the sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of the target sequence) bring. All or part of the wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence, or about 20, 26 , 32, 45, 48, 54, 63, 67, 85, or more nucleotides) may also comprise or consist of a tracr sequence functional to the guide sequence, e.g., along at least a portion of the tracr sequence. can form part of a CRISPR complex, such as by hybridization to all or part of the tracr mate sequence linked to the . The tracr sequence is sufficiently complementary to the tracr mate sequence to hybridize and participate in formation of the CRISPR complex, e.g. Have 70%, 80%, 90%, 95% or 99% sequence complementarity.

Introduction of one or more vectors driving expression of one or more elements of the CRISPR system into the cell such that expression of those elements of the CRISPR system results in the formation of a CRISPR complex at one or more target sites. can be commanded. Components can also be delivered to cells as proteins and/or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. The Cas enzyme can be a target gene under the control of a regulatory alternative splicing event disclosed herein as a chimeric target gene minigene or as a target gene for a chimeric minigene transactivator. A gRNA can be under the control of a constitutive promoter.

Alternatively, any CRISPR system in which two or more elements expressed from the same or different regulatory elements are combined in one vector and one or more additional vectors are not included in the first vector. components may be provided. A vector may contain one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning sites"). In some embodiments, one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors. By using multiple different guide sequences, a single expression construct can be used to target CRISPR activity to multiple corresponding target sequences within a cell.

The vector can include regulatory elements operably linked to an enzyme coding sequence that encodes a CRISPR enzyme such as the Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1 , Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx15, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified forms thereof. These enzymes are known. For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database as accession number Q99ZW2.

The CRISPR enzyme can be Cas9 (eg, from S. pyogenes or S. pneumonia). CRISPR enzymes can direct cleavage of one or both strands at the location of the target sequence, eg, within the target sequence and/or within the complementary strand of the target sequence. The vector can encode a mutated CRISPR enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence as compared to the corresponding wild-type enzyme. can. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (one that cleaves a single strand). Convert. In some embodiments, the Cas9 nickase may be used in combination with guide sequences, eg, two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination nicks both strands, allowing them to be used for induction of NHEJ or HDR.

In some embodiments, an enzyme-coding sequence encoding a CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. A eukaryotic cell can be of or derived from a particular organism, such as, but not limited to, a human, mouse, rat, rabbit, dog, or mammal, including non-human primates. Generally, codon-optimization refers to making at least one codon of a native sequence more frequent or less common in genes of that host cell while maintaining the native amino acid sequence, in order to enhance expression in the host cell of interest. Refers to the process of altering a nucleic acid sequence by replacing it with the most frequently used codon. Different species exhibit particular biases towards certain codons of particular amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the translation efficiency of messenger RNA (mRNA), and it is inter alia the characteristics of the codons translated and the specific transfer RNA (tRNA) molecule. is considered to depend on the availability of The predominance of a chosen tRNA in a given cell is generally a reflection of the codons most frequently used in peptide synthesis. Genes can therefore be adapted for optimal gene expression in a given organism based on codon optimization.

In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. . In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, or about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% , or more.

Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burrows- Algorithms based on Wheeler transforms (e.g. Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net).

A CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. A CRISPR enzyme fusion protein can include any additional protein sequences and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with one or more of the following activities: methylase. activity, demethylase activity, transcription activation activity, transcription repression activity, transcription terminator activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed. autofluorescent proteins including, but not limited to, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes are proteins or fragments of such proteins that bind to DNA molecules or to other cellular molecules such as, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA. The fusions can be to gene sequences encoding binding domain (DBD) fusions, GAL4A DNA binding domain fusions, herpes simplex virus (HSV) BP16 protein fusions, and the like. Additional domains that may form part of a fusion protein containing the CRISPR enzyme are described in US 20110059502, incorporated herein by reference.

D. Therapeutic Proteins Some embodiments relate to expression of recombinant proteins and polypeptides. In some aspects, proteins or polypeptides may be modified to increase serum stability. Thus, when this application refers to a function or activity of a "modified protein" or "modified polypeptide," it encompasses proteins or polypeptides that have additional advantages over, for example, unmodified proteins or polypeptides. It will be understood by those skilled in the art to do. It is specifically contemplated that aspects relating to "variant proteins" may also be implemented for "variant polypeptides" and vice versa.

A recombinant protein may possess amino acid deletions and/or substitutions. Thus, proteins with deletions, substitutions, and deletions and substitutions are modified proteins. In some embodiments, these proteins may further comprise inserted or added amino acids, eg, fusion proteins or proteins with linkers. A "modified deletion protein" may lack one or more residues of the native protein, but retain the specificity and/or activity of the native protein. A "modified deletion protein" may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is one in which amino acid residues have been deleted from at least one antigenic region, i.e., a region of the protein determined to be antigenic in a particular organism, e.g., in the organism to which the modified protein is administered. It is what we are doing.

Substitutional or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein to modify one or more properties of the polypeptide, particularly its effector functions and /or can be designed to modulate bioavailability. Substitutions may or may not be conservative, ie, replacements in which one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine to glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine changes, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine.

In addition to deletions or substitutions, variant proteins may possess insertions of residues. This typically involves adding at least one residue in the polypeptide. This may involve insertions of targeting peptides or targeting polypeptides or simple single residue insertions. Terminal additions called fusion proteins are described below.

The term "biologically functional equivalent" is well understood in the art and is further elaborated herein. Thus, a sequence in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to the amino acids of the reference polypeptide are , are included as long as the biological activity of the protein is maintained. A recombinant protein can, in certain aspects, be a biologically functional equivalent to the corresponding native protein.

Amino acid and nucleic acid sequences may contain additional residues, such as additional N-terminal or C-terminal amino acids, or 5′ or 3′ sequences, and even if that sequence is relevant for protein expression. It will also be understood to be essentially as described in one of the sequences disclosed herein, so long as it meets the above criteria, including, for example, maintenance of biological protein activity. The addition of terminal sequences may include, for example, various non-coding sequences flanking either the 5' or 3' portion of the coding region, or various internal sequences known to exist within the gene, i.e., introns. This is especially true for nucleic acid sequences that may contain

As used herein, a protein or peptide is generally a protein of greater than about 200 amino acids, up to the full-length sequence, translated from a gene; a polypeptide of greater than about 100 amino acids; and/or from about 3 to about 100 It refers to, but is not limited to, peptides of amino acids. For convenience, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein.

As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the residues of the protein or peptide are contiguous, with no non-amino acid sequences interrupting the sequence of amino acid residues. In another embodiment, the sequence may contain one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

Thus, the term "protein or peptide" encompasses amino acid sequences that contain at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

Certain aspects of the invention relate to fusion proteins. These molecules can have a therapeutic protein linked at the N-terminus or C-terminus to a heterologous domain. For example, fusions may also employ leader sequences from other species to allow recombinant expression of proteins in heterologous hosts. Other useful fusions are protein affinity tags, such as serum albumin affinity tags or six histidine residues, preferably cleavable, or immunologically active proteins such as antibody epitopes, to facilitate purification of the protein. including adding a valid domain. Non-limiting examples of affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST).

Methods of making fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by de novo synthesis of the entire fusion protein or by attachment of a DNA sequence encoding the heterologous domain and subsequent expression of the intact fusion protein.

The production of fusion proteins that restore the functional activity of the parent protein can be facilitated by joining the genes with bridging DNA segments encoding peptide linkers joined between the tandemly joined polypeptides. The linker is of sufficient length to allow proper folding of the resulting fusion protein.

III. Methods of Administration Viral vectors may, in some aspects, be administered directly (in vivo) to a patient, or may be used to treat cells in vitro or ex vivo and then administered to the patient. . The term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (eg, AAV vectors, retroviral vectors, lentiviral vectors), or other vehicle capable of manipulation by insertion or incorporation of nucleic acid. Vectors, such as viral vectors, are used to introduce/transfer nucleic acids into cells such that a nucleic acid sequence within the nucleic acid is transcribed and, if it encodes a protein, subsequently translated by the cell. can be used.

Any suitable cell or mammal can be administered or treated by the methods or uses described herein. Typically, a mammal in need of the methods described herein has or is suspected of expressing an aberrant protein or an aberrant protein associated with a disease state. Alternatively, the mammalian recipient may have a condition amenable to gene replacement therapy. As used herein, "gene replacement therapy" refers to the administration of exogenous genetic material encoding a therapeutic agent to a recipient, and the subsequent expression of the administered genetic material in situ. Thus, the phrase "conditions amenable to gene replacement therapy" includes genetic diseases (i.e., disease states resulting from one or more gene defects), acquired conditions (i.e., disease states not resulting from congenital defects). ), cancer and prophylactic processes (ie, prevention of disease or prevention of undesirable medical conditions). Accordingly, the term "therapeutic agent" as used herein refers to any agent or material that exerts a beneficial effect on a mammalian recipient. Thus, "therapeutic agent" encompasses both therapeutic and prophylactic molecules having nucleic acid or protein components.

Non-limiting examples of mammals include humans, non-human primates (such as apes, gibbons, chimpanzees, orangutans, monkeys, macaques), domestic animals (such as dogs and cats), agricultural animals (such as horses, cows, goats, sheep, pigs) and experimental animals (eg mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is human. In certain embodiments, the mammal is a non-rodent mammal (eg, humans, pigs, goats, sheep, horses, dogs, etc.). In certain embodiments, the non-rodent mammal is human. The mammal can be of any age or stage of development (eg, adult, teenager, child, infant or intrauterine mammal). Mammals can be male or female. In certain embodiments, the mammal is, for example, an animal model that has or expresses an aberrant protein or an aberrant protein associated with a disease state, or an animal model in which the protein is insufficiently expressed such that it causes the disease. such as animal disease models.

Mammals (subjects) to be treated with the methods or compositions described herein include adults (18 years of age or older) and children (under 18 years of age). Adults include the elderly. A typical adult is 50 years of age or older. Children range in age from 1-2 years or 2-4 years, 4-6 years, 6-18 years, 8-10 years, 10-12 years, 12-15 years, and 15-18 years. Children also include infants. Infants typically range in age from 1 to 12 months.

In certain embodiments, the method comprises administering to a mammal a plurality of viral particles, as described herein, to reduce the severity of one or more symptoms of a disease state, such as a neurodegenerative disease. Reduce, reduce, prevent, inhibit or delay the degree, frequency, progression or time of onset. In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to treat adverse symptoms of disease states, such as neurodegenerative diseases. In certain embodiments, the method administers a plurality of viral particles to a mammal to stabilize, delay, or prevent worsening or progression or reversal of disease states and adverse symptoms, such as neurodegenerative diseases. Including stages.

In certain embodiments, the method comprises administering a plurality of viral particles to the central nervous system or a portion thereof, as described herein, to treat one or more disease states, such as neurodegenerative disease. reduce, reduce, prevent, inhibit, or at least about 5 to about 10 days, about 10 to about 25 days, about 25 to about Delay for 50 days, or about 50 to about 100 days.

In certain embodiments, the symptoms or adverse effects are early, intermediate or late symptoms, behavioral, personality or speech symptoms, swallowing, movements, seizures, tremors or restlessness, ataxia, and/or Includes cognitive symptoms such as memory and the ability to organize.

IV. Pharmaceutical Compositions The terms "pharmaceutically acceptable" and "physiologically acceptable" as used herein refer to one or more routes of administration, in vivo delivery or in vivo contact. means any biologically acceptable composition, formulation, liquid or solid, or mixture thereof. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g. can be administered to a subject without causing any biological effect. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions can be sterile. Such pharmaceutical formulations and compositions can be used, for example, in administering viral particles to a subject.

Such formulations and compositions are suitable for pharmaceutical administration or for in vivo contact or delivery, in solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g. oil-in-water or in oil). Aqueous forms), suspensions, syrups, elixirs, dispersion media and suspending media, coatings, isotonicizing and absorption enhancers or delayers. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Supplementary active compounds such as preservatives, antibacterial, antiviral and antifungal agents can also be incorporated into the formulations and compositions.

A pharmaceutical composition typically contains a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of harmful antibodies in an individual to whom the composition is administered and that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. It contains pharmaceutically acceptable salts, for example mineral salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and acetates, propionates, malonates, benzoates, etc. can include salts of organic acids of Furthermore, auxiliary substances such as surfactants, wetting or emulsifying agents, pH buffering substances and the like can be present in such media.

A pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery described herein or known to those skilled in the art. Pharmaceutical compositions, therefore, include carriers, diluents or excipients suitable for administration or delivery by various routes.

Pharmaceutical forms suitable for injection of viral particles are sterile aqueous solutions or dispersions adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. body can be mentioned. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture, use and storage. Liquid carriers or media can be solvents or liquid dispersion media including, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. can. Proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Tonicity agents such as sugars, buffers or salts (eg sodium chloride) can be included. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents that delay absorption, such as aluminum monostearate and gelatin.

A solution or suspension of viral particles can optionally include one or more of the following components: sterile diluents such as water for injection, saline solutions such as phosphate buffered saline (PBS). , artificial CSF, surfactants, fixed oils, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols), glycerin or other synthetic solvents, antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid. antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffering agents, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as chloride. sodium or dextrose.

Pharmaceutical formulations, compositions and delivery systems suitable for the compositions, methods and uses of the present invention are known in the art (e.g. Remington: The Science and Practice of Pharmacy (2003) 20^th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18^th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12^th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11^th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

Virus particles and compositions thereof can be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the individual to be treated, each unit calculated to produce the desired therapeutic effect. It contains a predetermined amount of active compound together with the required pharmaceutical carrier. The dosage unit form will depend on the number of viral particles deemed necessary to produce the desired effect. The required amount can be formulated in a single dose or in multiple dosage units. Doses may be adjusted for appropriate viral particle concentrations, optionally combined with anti-inflammatory agents, and packaged for use.

In one embodiment, the pharmaceutical composition is in a therapeutically effective amount, i.e., an amount sufficient to reduce or ameliorate the symptoms or adverse effects of the disease state in question, or an amount sufficient to provide the desired benefit. contains sufficient genetic material to provide

As used herein, "unit dosage form" refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing one or more doses of optionally containing a pharmaceutical carrier (excipient, diluent, vehicle or filler) in a predetermined amount calculated to produce a desired effect (e.g., prophylactic or therapeutic effect) when administered once do. Unit dosage forms can be, for example, in ampoules and vials, which can contain a liquid composition, or the composition in a freeze-dried or lyophilized state, prior to in vivo administration or delivery, for example, with the addition of a sterile liquid carrier. be able to. Individual unit dosage forms can be included in a multi-dose kit or container. Thus, for example, viral particles, and pharmaceutical compositions thereof, can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.

Viral particle-containing formulations typically contain an effective amount, which is readily determined by one skilled in the art. Viral particles can typically range from about 1% to about 95% (w/w) of the composition, or higher if appropriate. The amount administered will depend on factors such as the age, weight and health of the mammal or human subject being considered for treatment. Effective dosages can be established by one of ordinary skill in the art through routine trials establishing dose-response curves.

V. DEFINITIONS The terms "polynucleotide", "nucleic acid" and "transgene" are used herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and polymers thereof. Used interchangeably to refer to Polynucleotides include genomic DNA, cDNA and antisense DNA, as well as spliced or unspliced mRNA, rRNA, tRNA and inhibitory DNA or inhibitory RNA (RNAi, e.g., small or short hairpin (sh) RNA , microRNAs (miRNAs), small or short interfering (si) RNAs, trans-splicing RNAs or antisense RNAs). Polynucleotides can include natural, synthetic, and intentionally modified or modified polynucleotides (eg, variant nucleic acids). A polynucleotide can be single-, double-, or triple-stranded, linear or circular, and can be of any suitable length. In discussing polynucleotides, the sequence or structure of a particular polynucleotide herein may be described according to the convention of writing the sequence in the 5' to 3' direction.

A nucleic acid that encodes a polypeptide often includes an open reading frame that encodes the polypeptide. Certain nucleic acid sequences also contain degenerate codon substitutions, unless otherwise indicated.

A nucleic acid can include one or more expression control elements or regulatory elements operably linked to an open reading frame, which regulatory element or elements are encoded by the open reading frame in a mammalian cell. It is configured to direct transcription and translation of a polypeptide. Non-limiting examples of expression control/regulatory elements include transcription initiation sequences (eg, promoters, enhancers, TATA boxes, etc.), translation initiation sequences, mRNA stability sequences, poly A sequences, secretory sequences, and the like. Expression control/regulatory sequences can be obtained from the genome of any suitable organism.

A "promoter" is a term usually upstream (5') to a coding sequence that directs and/or controls expression of the coding sequence by providing recognition sites for RNA polymerase and other factors necessary for proper transcription. A nucleotide sequence that A pol II promoter contains a minimal promoter, a short DNA sequence composed of a TATA box and, optionally, other sequences that serve to specify the transcription initiation site, to which regulatory elements are added to control expression. be done. Type 1 pol III promoters have three cis-acting sequence elements downstream of the transcription initiation site: a) a 5' sequence element (A block); b) an intermediate sequence element (I block); c) a 3' sequence element (C blocks). Type 2 pol III promoters contain two essential cis-acting sequence elements downstream of the transcription start site: a) A box (5' sequence element); and b) B box (3' sequence element). Type 3 pol III promoters contain several cis-acting promoter elements upstream of the transcription start site, such as the conventional TATA box, proximal sequence element (PSE) and distal sequence element (DSE).

An "enhancer" is a DNA sequence capable of stimulating transcriptional activity and can be a promoter-specific or heterologous element that enhances the level of expression or the tissue specificity of expression. It can operate in either direction (5'→3' or 3'→5') and has the ability to function whether placed upstream or downstream of the promoter.

Promoters and/or enhancers may be derived in their entirety from a native gene, or be composed of different elements derived from different elements found in nature, or even be composed of synthetic DNA segments. A promoter or enhancer may contain DNA sequences involved in the binding of protein factors that modulate/control the effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.

Non-limiting examples of promoters include SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, e.g. Early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, pol II promoter, pol III promoter, synthetic promoter, hybrid promoter and the like. In addition, sequences derived from non-viral genes, such as the mouse metallothionein gene, are also considered useful herein. Exemplary constitutive promoters include the promoters of the following genes that encode certain constitutive or "housekeeping" functions, and other constitutive promoters known to those of skill in the art: hypoxanthine phosphoribosyltransferase ( HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, U6. In addition, many viral promoters function constitutively in eukaryotic cells. These include, among others, the early and late promoters of SV40, the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. In addition, sequences derived from intronic miRNA promoters, such as the miR107, miR206, miR208b, miR548f-2, miR569, miR590, miR566, and miR128 promoters, will also find use herein (e.g., Monteys et al., 2010). Thus, any of the constitutive promoters described above can be used to control transcription of heterologous gene inserts.

A "transgene" is used herein for convenience to refer to a nucleic acid sequence/polynucleotide that is or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as an inhibitory RNA or a gene encoding a polypeptide or protein, which is generally heterologous to the native AAV genomic sequence.

The term "transduction" refers to the introduction of a nucleic acid sequence into a cell or host organism by a vector (eg, a viral particle). Thus, introduction of a transgene into a cell by a viral particle can be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. When an introduced transgene is integrated into the nucleic acid (genomic DNA) of a recipient cell or organism, it is allowed to be stably maintained in that cell or organism and the progeny of the recipient cell or organism further transmitted or passed on to cells or progeny organisms. Finally, the introduced transgene may exist extrachromosomally or only transiently in the recipient cell or recipient host organism. A "transduced cell" is therefore a cell into which a transgene has been introduced by transduction. A "transduced" cell is thus a cell into which a transgene has been introduced or its progeny. Transduced cells are allowed to grow, transcribe the transgene, and express the encoded inhibitory RNA or protein. For gene therapy uses and methods of gene therapy, the transduced cells can be in a mammal.

A transgene under the control of an inducible promoter will be expressed only in the presence of the inducing agent, or will be more strongly expressed in the presence of the inducing agent (e.g., transcription under the control of the metallothionein promoter is expressed in the presence of certain metal ions increase significantly). Inducible promoters contain responsive elements (REs) that stimulate transcription when the respective inducer is bound. Examples are REs for serum factors, steroid hormones, retinoic acid and cyclic AMP. A promoter containing a particular RE can be chosen to obtain an inducible response, and optionally the RE itself may be ligated to a different promoter to confer inducibility on the recombinant gene. Thus, by choosing an appropriate promoter (constitutive or inducible, strong or weak), it is possible to control both the presence and expression levels of a polypeptide in genetically modified cells. If the gene encoding the polypeptide is under the control of an inducible promoter, delivery of the polypeptide in situ can be accomplished by exposing the genetically modified cell in situ to conditions that permit transcription of the polypeptide, e.g. is triggered by intraperitoneal injection of specific inducers of inducible promoters that control the transcription of . For example, in situ expression by genetically modified cells of polypeptides encoded by genes under the control of the metallothionein promoter can be achieved by contacting the genetically modified cells in situ with a solution containing an appropriate (i.e., inducible) metal ion. enhanced by

A nucleic acid/transgene is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. Nucleic acids/transgenes encoding RNAi or polypeptides or nucleic acids that direct expression of the polypeptides may contain inducible promoters or tissue-specific promoters to control transcription of the encoded polypeptides. A nucleic acid operably linked to an expression control element can also be referred to as an expression cassette.

In certain embodiments, the methods and uses described herein employ CNS-specific or inducible promoters, enhancers, and the like. Non-limiting examples of CNS-specific promoters include those isolated from the genes for myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE). Non-limiting examples of inducible promoters include DNA responsive elements to ecdysone, tetracycline, hypoxia and IFN.

In certain embodiments, the expression control element comprises a CMV enhancer. In certain embodiments, the expression control element comprises the β-actin promoter. In certain embodiments, the expression control element comprises the chicken β-actin promoter. In certain embodiments, the expression control elements include the CMV enhancer and the chicken β-actin promoter.

As used herein, the terms "modify" or "variant" and grammatical variations thereof mean that a nucleic acid, polypeptide or subsequence thereof deviates from a reference sequence. Thus, modified and variant sequences may have substantially the same, greater, or lesser expression, activity or function than a reference sequence, but at least have the activity or function of the reference sequence. partially retained. A particular type of variant is a mutant protein, which refers to a protein encoded by a gene that has mutations, eg, missense or nonsense mutations.

A "nucleic acid" variant or "polynucleotide" variant refers to a modified sequence that alters the gene as compared to the wild type. A sequence can be genetically modified without altering the encoded protein sequence. Alternatively, sequences can be genetically modified to encode variant proteins. A nucleic acid or polynucleotide variant is codon-modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as a wild-type protein sequence, and to encode a variant protein. It can also refer to combination sequences that are codon-modified. For example, some codons of such nucleic acid variants are changed so as not to change the amino acids of the protein encoded by it, some codons of the nucleic acid variant are changed so that the It will change the amino acid of the protein.

The terms "protein" and "polypeptide" are used interchangeably herein. The "polypeptides" encoded by the "nucleic acids" or "polynucleotides" or "transgenes" disclosed herein include partial or full-length native sequences, as well as naturally occurring wild-type and functional polymorphic proteins. , functional subsequences (fragments) thereof, as well as sequence variants thereof, so long as the polypeptide retains some degree of function or activity. Thus, in the methods and uses of the invention, such polypeptides encoded by the nucleic acid sequences are deficient in endogenous proteins that are deficient, or whose activity, function or expression is deficient in the mammal being treated. , need not be identical to the endogenous protein that is missing or absent.

Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (eg, about 1 to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000, or more nucleotides or residues).

Examples of amino acid alterations are conservative amino acid substitutions or deletions. In certain embodiments, a modified or variant sequence retains at least part of the function or activity of the unmodified sequence (eg, wild-type sequence).

Another example of an amino acid modification is a targeting peptide that is introduced into the viral particle capsid protein. Peptides have been identified that target recombinant viral vectors to the central nervous system, including different brain regions.

A recombinant virus so modified may preferentially bind to one type of tissue (eg, CNS tissue) over another type of tissue (eg, liver tissue). In certain embodiments, recombinant viruses carrying modified capsid proteins can "target" brain vascular epithelial tissue by binding at higher levels than comparable unmodified capsid proteins. For example, recombinant viruses with modified capsid proteins can bind to cerebrovascular epithelial tissue at 50% to 100% higher levels than unmodified recombinant viruses.

A "nucleic acid fragment" is a portion of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) is the genetic material in most organisms, while ribonucleic acid (RNA) is responsible for transferring the information contained within DNA to proteins. Fragments and variants of the disclosed nucleotide sequences and proteins or partial length proteins encoded thereby are also encompassed by the present invention. By "fragment" or "portion" is meant a full-length or less than full-length nucleotide sequence encoding a polypeptide or protein or amino acid sequence of a polypeptide or protein. In certain embodiments, the fragment or portion is biologically functional (i.e., 5%, 10%, 15%, 20%, 25%, 30%, 35% of wild-type activity or function). , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%).

A "variant" of a molecule is a sequence that is substantially similar to that of the native molecule. In the case of nucleotide sequences, variants include sequences that encode the same amino acid sequence as the native protein due to the degeneracy of the genetic code. Such naturally occurring allelic variants can be identified using molecular biology techniques such as, for example, the polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include nucleotide sequences of synthetic origin, such as those generated using site-directed mutagenesis, which encode native proteins, as well as those which encode polypeptides having amino acid substitutions. Generally, a nucleotide sequence variant of the invention will be at least 40%, 50%, 60% or 70% relative to the native (endogenous) nucleotide sequence, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, generally at least 80%, such as 81% to 84%, at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity. In certain embodiments, the variant is biologically functional (i.e., has 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% of the wild-type activity or function). %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%).

"Conservative substitutions" of a particular nucleic acid sequence refer to nucleic acid sequences that encode identical or essentially identical amino acid sequences. Because the genetic code is degenerate, any given polypeptide is encoded by multiple functionally identical nucleic acids. For example codons CGT, CGC, CGA, CGG, AGA and AGG all encode the amino acid arginine. Thus, wherever a codon specifies arginine, the codon can be changed to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations," which are one species of "conservatively modified variations." Any nucleic acid sequence described herein which encodes a polypeptide represents all possible silent variations, unless otherwise noted. Those skilled in the art will recognize that each codon in a nucleic acid (except for ATG, which is usually the only methionine codon) can be altered to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

The term "substantial identity" of a polynucleotide sequence means that the polynucleotide exhibits at least 70%, 71%, 72% identity when compared to a reference sequence using one of the described alignment programs with standard parameters. %, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, Sequences with 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity is meant to contain It is understood that these values can be appropriately adapted, taking into account codon degeneracy, amino acid similarity, reading frame position, etc., to determine the corresponding identities of proteins encoded by two nucleotide sequences. , will be understood by those skilled in the art. For these purposes, substantial amino acid sequence identity usually means at least 70%, at least 80%, 90%, or even at least 95% sequence identity.

The term "substantial identity" with respect to a polypeptide means that the polypeptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76% identity to a reference sequence over a specified window of comparison. %, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91 %, 92%, 93% or 94%, or even 95%, 96%, 97%, 98% or 99% sequence identity. A measure of the identity of two polypeptide sequences is that one polypeptide reacts immunologically with antibodies raised against the other polypeptide. Thus, one polypeptide is identical to another polypeptide, for example, where the two polypeptides differ only by conservative substitutions.

The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent undesirable physiological changes or disorders, such as the occurrence, progression or exacerbation of disorders. , to prevent, inhibit, reduce or decrease. For the purposes of the present invention, beneficial or desirable clinical results include alleviation of symptoms, reduction in extent of disease, stabilization of symptoms or adverse effects of disease, whether detectable or undetectable. slowing or slowing disease progression, amelioration or alleviation of disease status, and remission (whether partial or complete). never "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those who already have the condition or disorder as well as those who have a predisposition (eg, as determined by genetic assays).

VI. Kits The present invention provides kits with packaging and one or more components therein. Kits typically include a label or package insert that includes a description of the components contained therein or instructions for in vitro, in vivo, or ex vivo use of the components contained therein. A kit can include a collection of such components, such as nucleic acids, recombinant vectors, and/or viral particles.

Kit refers to a physical structure that houses one or more components of the kit. The packaging is capable of maintaining sterility of the components and is made of materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, vials, tubes, etc.) be able to.

A label or package insert can include the identity of one or more of the components contained therein, the dosage, the clinical pharmacology of the active ingredient, such as mechanism of action, pharmacokinetics and pharmacodynamics. The label or package insert can include information identifying the manufacturer, lot number, place and date of manufacture, and expiration date. The label or package insert may include information identifying manufacturer information, lot number, place of manufacture and date of manufacture. The label or package insert can include information regarding the disease for which the components of the kit can be used. The label or package insert can include instructions for the clinician or subject to use one or more of the components of the kit in a method, use or treatment protocol or regimen. Instructions can include dosage, frequency or duration of dosing, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimens described herein.

The label or package insert can include information regarding the benefits that the component may provide, such as prophylactic or therapeutic benefits. The label or package insert may include information regarding potentially adverse side effects, complications or reactions, e.g., warning the subject or clinician regarding circumstances in which it may not be appropriate to use a particular composition. can. Adverse side effects or complications may also occur if the subject has taken, will take, or is currently taking one or more other medications that may be incompatible with the composition, or if the subject may also occur if a patient has previously received, will receive, or is currently receiving another treatment protocol or therapeutic regimen that is considered incompatible with the composition, the instructions shall also include information regarding such incompatibilities. can be included.

Labels or package inserts are "printed material", either separate or attached to a component, kit or packaging (e.g. a box), or to an ampoule, tube or vial containing a component of a kit, e.g. Including paper or cardboard. A label or package insert may be a computer readable medium such as a printed label with bar code, disc, optical disc such as CD- or DVD-ROM/RAM, DVD, MP3, or electronic storage medium such as RAM and ROM, or hybrids thereof. , such as magnetic/optical storage media, flash memory, hybrid and memory type cards.

VII. Examples The following examples are provided to demonstrate preferred embodiments of the invention. As will be appreciated by those of skill in the art, the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus the practice thereof. can be regarded as constituting a preferred form for However, one of ordinary skill in the art, in light of the present disclosure, may make many changes to the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the invention. you will understand that

Example 1 - Identification of AAV variants that target the brain parenchyma
AAV1, AAV2, and AAV9 capsids were used as starting platforms to develop advanced barcoded AAV libraries. AAV1, AAV2, and AAV9 peptide display libraries were generated by inserting random sequences at position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, and position 588 of the AAV9 capsid, respectively (Figure 1). This library had a diversity of 1×10 ⁷ unique clones (FIG. 3).

To test the utility of the library, a pilot study was performed using bench-grade (low titer, low purity) capsid-modified AAV2. The AAV2 library was injected intravenously into two C57BL/6 mice at 8×10 ¹⁰ vector genomes per animal. After 72 hours, the cerebral cortex, cerebellum, and spinal cord were dissected. Of note, the heart, skeletal muscle, and diaphragm were harvested separately to determine muscle tropism. Viral genomic DNA was isolated and the recovered random oligonucleotide sequences were amplified by PCR. PCR products from brain were pooled to generate a second round library, which was injected into two mice at 4×10 ¹⁰ vector genomes per animal. After the second injection, the vector genome was recovered as before and subjected to NexGen sequencing along with the starting library and first round tissue. To test whether sequences demonstrating enrichment in brain tissue could indeed allow AAV2 to reach the brain, individual hits were cloned into the AAV2 capsid packaging plasmid to generate eGFP-expressing AAV2. . A bench-grade vector was generated and mice were injected with 3×10 ¹⁰ AAV2-based capsid-modified viral vector genomes. After 4 weeks, eGFP fluorescence was seen in the brain even for these low titer variants.

Using these advanced barcoded AAV libraries, we identified AAV variants capable of targeting different primate brain structures in non-human primates. AAV1, AAV2, and AAV9 libraries were delivered to one non-human primate by intracerebroventricular injection (Fig. 2). Seventy-two hours after injection, brain regions were microdissected for viral DNA isolation and AAV DNA was amplified by PCR. Products were pooled and used for packaging a second round library, which was injected into additional NHPs. Brain regions were then microdissected 12 days after injection. After two rounds of panning, the vector genome was recovered and subjected to next generation sequencing. Specifically, genomic DNA extracted from round 1 and round 2 tissues was PCR amplified to generate Illumina amplicon sequencing libraries at the vector barcode locations. The resulting libraries were pooled and run on an Illumina HiSeq 4000 single lane using 100 bp single-ended read chemistry. To illustrate the utility of this approach, several target regions were tested as examples: ependyma, meninges and cerebellum. In general, the sequences that direct AAVx to the ependyma, meninges, and cerebellum were distinct and varied among the various serotypes.

Round-over-round enrichment graph (Figure 4) and heatmaps (Figures 5 and 6) of the following tissues: brain stem, caudate nucleus, cerebellar cortex (Figure 5), cerebral cortex, ependyma, globus pallidum, hippocampus, meninges, The optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, and thalamus were made. These illustrate the enrichment of the indicated barcodes at baseline (Round 0) and after rounds 1 and 2 of in vivo passaging in rhesus monkeys. To generate these, fastq result files for each tissue and round combination were processed using a custom Python script designed to extract and quantify the unique barcode configurations observed at the DNA level. A custom R script was used to calculate the percentage of barcodes present in each sample and convert DNA barcodes to amino acid barcodes. Table 1 corresponds to samples treated with AAV1-derived libraries; Table 2 corresponds to tissues treated with AAV2-derived libraries; Table 3 corresponds to samples treated with AAV9-derived libraries. Top hits from these three libraries were selected and assembled into a validation library containing barcodes from 50 (AAV1), 58 (AAV2) and 30 (AAV9). This validation library was delivered to additional rhesus monkeys by ICV injection. Tissues were collected and processed again to facilitate recovery of barcode abundance by deep sequencing. Barcode abundance was assessed in harvested tissues and input virus library. Enrichment values for each barcode were calculated relative to their abundance in the input virus library. The relative enrichment values obtained are robust indicators of vector performance among the various tissues evaluated, facilitating identification of broad and specific AAV vector variants (Figure 7A-C).

To validate the identified cell type specificity, AAV9-1999 (KGGGFHG; has a targeting peptide sequence of SEQ ID NO: 110) was selected for in vivo validation. The eGFP expression construct was packaged into AAV9-1999 driven by the CAG promoter. Five-year-old female rhesus monkeys were administered AAV9-1999 1.5E13 vg by ICV injection into the left lateral ventricle. Brains were collected 30 days after injection for histological analysis. Cerebellar slices were H&E stained to delineate the transduction pattern of AAV9-1999 (Figure 8). Cochleae were also collected from this animal and surprisingly had strong transduction of hair cells. Additionally, AAV9-1999 and AAV9 capsids containing eGFP constructs were delivered to C57BL/6 p0 mouse pups by ICV injection at 1E10 vg per hemisphere. After 21 days, mice were perfused. Whole mount brain (Figure 9A), 40 µm whole brain sagittal section (Figure 9B), 40 µm S1 cortex section (Figure 9C, left), 40 µm hippocampal section (Figure 9C, middle), 40 µm cerebellar sagittal section (Figure 9C, middle). FIG. 9C, right), and 40 μm lumbar cord coronal sections (FIG. 9D) were imaged for eGFP fluorescence signal. AAV9-1999 injected into Bl/6 neonatal mouse pups showed more ubiquitous expression than dose-matched injections of AAV9.

A mixture of 4 modified AAVs per adult rhesus monkey: AAV9 with RGDLQWV (SEQ ID NO: 113) targeting peptide sequence and mTAGBFP2 tag; AAV1 with ERDRTRG (SEQ ID NO: 21) targeting peptide sequence as mTFP1; GRGAPGG (SEQ ID NO: 21) ID NO: 80) AAV2 with targeting peptide sequence and mNG tag; and DDPSARR (SEQ ID NO: 53) AAV2 with targeting peptide sequence and mRuby3 tag were injected. Equal volumes of virus were directly mixed to achieve the final total dose of each as follows.
AAV9.RGDL mTagBFP2 6.13E12 all vg
AAV1.ERDR mTFP1 1.23E13 all vg
AAV2.GRGA mNG 8.8E12 all vg
AAV2.DDPS mRuby3 1.32E13 all vg

Brains were harvested 30 days after injection for fluorescence imaging. Lateral ventricular (FIG. 10A), fourth ventricular (FIG. 10B), and meningeal (FIG. 10C) sections were imaged for mTagBFP2, mTFP2, mNG, and mRuby3 fluorescence signals.

Additional experiments were performed by injecting AAV9-1999 into the cochlea of rhesus monkeys. Based on the results of cochlear transduction, animals were administered AAV9-1999 into the lateral ventricle. A single animal received AAV9-1999 3E11 vg injected directly into its cochlear window at a fenestration (FIGS. 11A-C).

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, various steps or orders of steps of the methods and methods described herein may be made without departing from the concept, spirit and scope of the present invention. It will be clear to those skilled in the art that variations may apply. More specifically, certain agents, both chemically and physiologically related, can be used in place of the agents described herein and achieve the same or similar results. It would be obvious. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Claims (196)

A modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide, said targeting peptide allowing the viral vector containing said modified AAV capsid protein to be targeted to different organs or different brain structures, comprising 3-10 amino acids. The modified AAV capsid protein that is long. wherein said modified AAV capsid protein is a modified AAV9 capsid protein having a sequence at least 95% identical to SEQ ID NO: 143, said targeting peptide is SEQ ID NO: 110, and said different brain structure is Brain stem, caudate nucleus, cerebellum, cochlea (ear), cortex, cerebral cortex, deep cerebellar nucleus, ependym, globus pallidus, hippocampus, meninges, motor cortex, optic nerve, prefrontal cortex, putamen, spinal cord, substantia nigra, 2. The modified AAV capsid protein of claim 1, which is the subthalamic nucleus, temporal cortex, thalamus, or visual cortex. 2. The modified AAV capsid protein of claim 1, which is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein. 2. The modification of claim 1, wherein said modified AAV capsid protein is derived from the AAV1 capsid protein (see SEQ ID NO: 138) and said targeting peptide is inserted after residue 590 of said AAV1 capsid protein. AAV capsid protein that has been modified. 5. The modified AAV capsid protein of claim 4, wherein said targeting peptide is flanked by linker sequences, said linker sequences on either side of said targeting peptide being 2 or 3 amino acids in length. 6. The modified AAV capsid protein of claim 5, wherein said linker sequence is SSA N-terminal to said targeting peptide and AS C-terminal to said targeting peptide. 7. The modified AAV capsid protein of claim 6, wherein said modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:141. 2. The modification of claim 1, wherein said modified AAV capsid protein is derived from the AAV2 capsid protein (see SEQ ID NO: 139) and said targeting peptide is inserted after residue 587 of said AAV2 capsid protein. AAV capsid protein that has been modified. 9. The modified AAV capsid protein of claim 8, wherein said targeting peptide is flanked by linker sequences, said linker sequences on either side of said targeting peptide being 2 or 3 amino acids in length. 10. The modified AAV capsid protein of claim 9, wherein said linker sequence is AAA N-terminal to said targeting peptide and AA C-terminal to said targeting peptide. 11. The modified AAV capsid protein of claim 10, wherein said modified AAV2 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:142. 2. The modification of claim 1, wherein said modified AAV capsid protein is derived from the AAV9 capsid protein (see SEQ ID NO: 140) and said targeting peptide is inserted after residue 588 of said AAV9 capsid protein. AAV capsid protein that has been modified. 13. The modified AAV capsid protein of claim 12, wherein said targeting peptide is flanked by linker sequences, said linker sequences on either side of said targeting peptide being 2 or 3 amino acids in length. 14. The modified AAV capsid protein of claim 13, wherein said linker sequence is AAA N-terminal to said targeting peptide and AS C-terminal to said targeting peptide. 15. The modified AAV capsid protein of claim 14, wherein said modified AAV9 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:143. 2. The modified AAV capsid of claim 1, wherein said target peptide comprises a sequence up to 10 amino acids long and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-137 or 144 in said sequence. protein. 17. The modified AAV capsid protein of claim 16, wherein said targeting peptide is 7 amino acids long. 3. The different brain structures are brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidum, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. Modified AAV capsid protein according to any one of 1-17. 19. The modified brain structure of claim 18, wherein said different brain structure is the brainstem, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 1-9. AAV capsid protein. said different brain structure is the caudate nucleus, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is SEQ ID NO: 1, 3, 5, 7, 10-16, 25 19. The modified AAV capsid protein of claim 18, which is selected from , 26, 32, and 144. said different brain structure is the cerebellar cortex, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 1, 3, 4, 9, and 17-21 19. The modified AAV capsid protein of claim 18, wherein the AAV capsid protein is said different brain structure is the cerebral cortex, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 1, 3, 5, 12, and 21-26 19. The modified AAV capsid protein of claim 18, wherein the AAV capsid protein is said different brain structure is the ependyma, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is SEQ ID NO: 2-4, 7, 9, 21, 22, 27, and 19. The modified AAV capsid protein of claim 18, selected from 28. said different brain structure is globus pallidum, said modified AAV capsid protein is modified AAV1 capsid protein, and said targeting peptide is SEQ ID NO: 3, 5, 12, 14, 16, 21, 22 , and 29-31. said different brain structure is the hippocampus, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 1-4, 7, and 32-34; 19. The modified AAV capsid protein of claim 18. said different brain structure is meninges, said modified AAV capsid protein is modified AAV1 capsid protein, and said targeting peptide is SEQ ID NO: 3, 5, 7, 9, 12, 21, and 35 19. The modified AAV capsid protein of claim 18, selected from -37. said different brain structure is the optic nerve, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is SEQ ID NO: 2, 3, 7, 14-16, 21, 31, and 19. The modified AAV capsid protein of claim 18, selected from 38. said different brain structure is the putamen, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is SEQ ID NO: 3, 4, 12, 13, 21, 30, and 39 19. The modified AAV capsid protein of claim 18, selected from -42. said different brain structure is the spinal cord, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is SEQ ID NO: 2-4, 7, 9, 21, 32, 33, and 19. The modified AAV capsid protein of claim 18, selected from 43. said different brain structure is the substantia nigra, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is selected from SEQ ID NO: 2, 3, 9, 44, and 45 19. The modified AAV capsid protein of claim 18. said different brain structure is the subthalamic nucleus, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is SEQ ID NO: 2-4, 12, 16, 30, 46, and 19. The modified AAV capsid protein of claim 18, selected from 47. said different brain structure is the thalamus, said modified AAV capsid protein is a modified AAV1 capsid protein, and said targeting peptide is SEQ ID NO: 1, 2, 8, 12, 21, 28, and 48- 19. The modified AAV capsid protein of claim 18, selected from 51. 19. The modified brain structure of claim 18, wherein said different brain structure is the brainstem, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 52-60. AAV capsid protein. Claim 18, wherein said different brain structure is the caudate nucleus, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 59 and 61-69. A modified AAV capsid protein as described. said different brain structure is the cerebellar cortex, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 56, 58, 60, and 70-75 19. The modified AAV capsid protein of claim 18. said different brain structure is the cerebral cortex, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is SEQ ID NO: 53, 58, 60, 62, 63, 66, and 76 19. The modified AAV capsid protein of claim 18, selected from -79. said different brain structure is ependyma, said modified AAV capsid protein is modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 53, 60, 62, 63, 66, 74-77, and 19. The modified AAV capsid protein of claim 18, selected from 80. said different brain structure is the globus pallidum, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 60, 75, and 81-87; 19. The modified AAV capsid protein of claim 18. said different brain structure is the hippocampus, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is SEQ ID NO: 53, 55, 58, 60, 63, 76, 79, 88 , and 89. said different brain structure is meninges, said modified AAV capsid protein is modified AAV2 capsid protein, and said targeting peptide is SEQ ID NO: 58, 60, 66, 73, 76, 80, and 90 19. The modified AAV capsid protein of claim 18, selected from -93. said different brain structure is the optic nerve, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is SEQ ID NO: 53, 54, 57, 58, 60, 75, 79, 87 19. The modified AAV capsid protein of claim 18, which is selected from , 88, and 94. said different brain structure is the putamen, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 55, 59, 60, 61, and 95-100 19. The modified AAV capsid protein of claim 18, wherein the AAV capsid protein is said different brain structure is the spinal cord, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is SEQ ID NO: 53, 58-61, 63, 77, 88, 95, and 19. The modified AAV capsid protein of claim 18, selected from 101. said different brain structure is the substantia nigra, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is SEQ ID NO: 52, 53, 57, 58, 75, 76, 87, 19. The modified AAV capsid protein of claim 18, selected from 102, and 103. said different brain structure is the subthalamic nucleus, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is SEQ ID NO: 57, 58, 60, 75, 79, 87, 88 19. The modified AAV capsid protein of claim 18, which is selected from , 102, 104, and 105. said different brain structure is the thalamus, said modified AAV capsid protein is a modified AAV2 capsid protein, and said targeting peptide is SEQ ID NO: 52, 55, 56, 74, 85, 88, and 106 to 19. The modified AAV capsid protein of claim 18, selected from 109. 19. The modified brain structure of claim 18, wherein said different brain structure is the brainstem, said modified AAV capsid protein is a modified AAV9 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 110-117. AAV capsid protein. said different brain structure is the caudate nucleus, said modified AAV capsid protein is a modified AAV9 capsid protein, and said targeting peptide is from SEQ ID NOs: 110, 113, 115, 116, and 118-121 19. The modified AAV capsid protein of claim 18, selected. said different brain structure is the cerebellar cortex, said modified AAV capsid protein is a modified AAV9 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 110, 111, 113, 119, and 122-125 19. The modified AAV capsid protein of claim 18, wherein the AAV capsid protein is said different brain structure is the cerebral cortex, said modified AAV capsid protein is a modified AAV9 capsid protein, and said targeting peptide is SEQ ID NO: 110, 111, 113, 114, 116, and 125-127 19. The modified AAV capsid protein of claim 18, selected from wherein the different brain structure is the ependyma, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NOs: 110, 111, 113, 118-120, and 128. 19. The modified AAV capsid protein of claim 18, wherein the modified AAV capsid protein is wherein said different brain structure is globus pallidum, said modified AAV capsid protein is modified AAV9 capsid protein, and said targeting peptide is from SEQ ID NO: 110-112, 114, 119, 120, and 129 19. The modified AAV capsid protein of claim 18, selected. said different brain structure is the hippocampus, said modified AAV capsid protein is a modified AAV9 capsid protein, and said targeting peptide is SEQ ID NO: 110, 111, 113, 116, 123, 125, 129, and 19. The modified AAV capsid protein of claim 18, selected from 130. said different brain structure is meninges, said modified AAV capsid protein is modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 110, 111, 113, 114, 118, 119, 122, 19. The modified AAV capsid protein of claim 18, selected from and 131. said different brain structure is the optic nerve, said modified AAV capsid protein is a modified AAV9 capsid protein, and said targeting peptide is from SEQ ID NO: 110, 111, 114, 115, 117, 129, and 132 19. The modified AAV capsid protein of claim 18, selected. said different brain structure is putamen, said modified AAV capsid protein is modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 110, 112, 113, 116, 123, 127, 133, 19. The modified AAV capsid protein of claim 18, which is selected from and 134. said different brain structure is the spinal cord, said modified AAV capsid protein is a modified AAV9 capsid protein, and said targeting peptide is SEQ ID NO: 110, 113, 119, 120, 122, 123, 128, and 19. The modified AAV capsid protein of claim 18, selected from 134. wherein said different brain structure is the substantia nigra, said modified AAV capsid protein is modified AAV9 capsid protein, and said targeting peptide is selected from SEQ ID NOs: 110-114, 117, and 129. 19. The modified AAV capsid protein of paragraph 18. said different brain structure is the subthalamic nucleus, said modified AAV capsid protein is a modified AAV9 capsid protein, and said targeting peptide is SEQ ID NO: 110, 111, 113, 119, 120, 122, 132 , and 135. said different brain structure is the thalamus, said modified AAV capsid protein is a modified AAV9 capsid protein, and said targeting peptide is from SEQ ID NO: 110, 112-114, 125, 133, 136, and 137 19. The modified AAV capsid protein of claim 18, selected. wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 1, and said different brain structure is brain stem, caudate nucleus, cerebellar cortex, cerebral cortex, hippocampus, or 8. The modified AAV capsid protein of claim 1 or 7, which is thalamus. wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 2, and said different brain structures are brainstem, ependyma, hippocampus, optic nerve, spinal cord, substantia nigra, hypothalamus 8. The modified AAV capsid protein of claim 1 or 7, which is nuclear or thalamic. wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 3, and said different brain structures are brain stem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, 8. The modified AAV capsid protein of claim 1 or 7, which is globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, or subthalamic nucleus. wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 4, and said different brain structure is brainstem, cerebellar cortex, ependyma, hippocampus, putamen, spinal cord, or 8. The modified AAV capsid protein of claim 1 or 7, which is the subthalamic nucleus. said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 5, and said different brain structure is brainstem, cerebral cortex, globus pallidus, or meninges; 8. The modified AAV capsid protein of claims 1 or 7. 8. The modified AAV of claim 1 or 7, wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 6, and said different brain structure is the brainstem. capsid protein. wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 7, and said different brain structures are brainstem, caudate nucleus, ependyma, hippocampus, meninges, optic nerve, 8. The modified AAV capsid protein of claim 1 or 7 which is spinal cord. 8. The modified AAV capsid protein of claim 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 8, and the different brain structure is the brainstem or thalamus. AAV capsid protein. wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 9, and said different brain structure is brainstem, cerebellar cortex, ependyma, meninges, spinal cord, or substantia nigra 8. The modified AAV capsid protein of claim 1 or 7, which is 8. The claim 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 10 or 11, and the different brain structure is the caudate nucleus. A modified AAV capsid protein. The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 12, and the different brain structures are the caudate nucleus, cerebral cortex, globus pallidus, meninges, and tunica. 8. The modified AAV capsid protein of claim 1 or 7, which is shell, subthalamic nucleus, or thalamus. 8. Claims 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 13, and the different brain structure is the caudate or putamen. modified AAV capsid proteins. 14. The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 14, and the different brain structure is the caudate nucleus, globus pallidum, or optic nerve. A modified AAV capsid protein according to 1 or 7. 8. The claim 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 15, and the different brain structure is the caudate nucleus or the optic nerve. A modified AAV capsid protein. wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 16, and the different brain structure is the caudate nucleus, globus pallidus, optic nerve, or subthalamic nucleus. 8. The modified AAV capsid protein of claim 1 or 7, wherein the modified AAV capsid protein is 1. The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is any one of SEQ ID NOs: 17-20, and the different brain structure is the cerebellar cortex. or a modified AAV capsid protein according to 7. wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 21, and said different brain structures are cerebellar cortex, cerebral cortex, ependyma, globus pallidum, meninges , optic nerve, putamen, spinal cord, or thalamus. 2. The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 22, and the different brain structure is the cerebral cortex, ependyma, or globus pallidus. or a modified AAV capsid protein according to 7. 1. The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is any one of SEQ ID NOs: 23-26, and the different brain structure is the cerebral cortex. or a modified AAV capsid protein according to 7. 8. The modified AAV of claim 1 or 7, wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 27, and said different brain structure is the ependyma. capsid protein. 8. The modified AAV capsid protein of claim 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 28, and the different brain structure is the ependyma or thalamus. AAV capsid protein. 8. The modified AAV capsid protein of claim 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 29, and the different brain structure is globus pallidus. AAV capsid protein. wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 30, and said different brain structure is globus pallidum, putamen, or subthalamic nucleus. 8. The modified AAV capsid protein of paragraphs 1 or 7. 8. The claim 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 31, and the different brain structure is the globus pallidus or the optic nerve. A modified AAV capsid protein. 8. The claim 1 or 7, wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 32 or 33, and said different brain structure is hippocampus or spinal cord. A modified AAV capsid protein. 8. The modified AAV of claim 1 or 7, wherein said modified AAV capsid protein is modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 34, and said different brain structure is hippocampus. capsid protein. 1. The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is any one of SEQ ID NOs: 35-37, and the different brain structure is the meninges. or a modified AAV capsid protein according to 7. 8. The modified AAV of claim 1 or 7, wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 38, and said different brain structure is the optic nerve. capsid protein. 1. The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is any one of SEQ ID NOs: 39-42, and the different brain structure is the putamen. or a modified AAV capsid protein according to 7. 8. The modified AAV of claim 1 or 7, wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 43, and said different brain structure is the spinal cord. capsid protein. 8. The modification of claim 1 or 7, wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is SEQ ID NO: 44 or 45, and said different brain structure is the substantia nigra. AAV capsid protein that has been modified. 8. The claim 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 46 or 47, and the different brain structure is the subthalamic nucleus. A modified AAV capsid protein. Claim 1 or wherein said modified AAV capsid protein is a modified AAV1 capsid protein, said targeting peptide is any one of SEQ ID NOs: 48-51, and said different brain structure is the thalamus, or 7. A modified AAV capsid protein according to 7. 12. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 52, and the different brain structure is the brainstem, substantia nigra or thalamus. A modified AAV capsid protein as described. wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 53, and said different brain structures are brainstem, cerebral cortex, ependyma, hippocampus, meninges, optic nerve, spinal cord , or substantia nigra. 12. The modified AAV capsid protein of claim 1 or 11, wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 54, and said different brain structure is brainstem or optic nerve. AAV capsid protein. 1. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 55, and the different brain structure is the brainstem, hippocampus, putamen, or thalamus. or the modified AAV capsid protein according to 11. 12. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 56, and the different brain structure is the brain stem, cerebellar cortex, or thalamus. A modified AAV capsid protein as described. wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 57, and said different brain structure is brainstem, optic nerve, substantia nigra, or subthalamic nucleus. 12. The modified AAV capsid protein of paragraphs 1 or 11. wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 58, and said different brain structures are brainstem, cerebellar cortex, cerebral cortex, hippocampus, meninges, optic nerve , spinal cord, substantia nigra, or subthalamic nucleus. wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 59, and said different brain structure is brain stem, caudate nucleus, putamen, or spinal cord. 12. The modified AAV capsid protein of paragraphs 1 or 11. wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 60, and said different brain structures are brainstem, cerebellar cortex, cerebral cortex, ependyma, globus pallidum, 12. The modified AAV capsid protein of claim 1 or 11, which is hippocampus, meninges, optic nerve, putamen, spinal cord, or subthalamic nucleus. 2. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 61, and the different brain structure is the caudate, putamen, or spinal cord. or the modified AAV capsid protein according to 11. 2. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 62, and the different brain structure is the caudate nucleus, the cerebral cortex, or the ependyma. or the modified AAV capsid protein according to 11. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 63, and the different brain structure is the caudate nucleus, cerebral cortex, ependyma, hippocampus, or spinal cord , the modified AAV capsid protein of claim 1 or 11. wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NOs: 64, 65, and 67-69, and the different brain structure is the caudate nucleus 12. The modified AAV capsid protein of claim 1 or 11, which is wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 66, and the different brain structure is the caudate nucleus, cerebral cortex, ependyma, or meninges; 12. The modified AAV capsid protein of claims 1 or 11. 1. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NOs: 70-72, and the different brain structure is the cerebellar cortex. or the modified AAV capsid protein according to 11. 12. The claim 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 73, and the different brain structure is the cerebellar cortex or meninges. A modified AAV capsid protein. 12. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 74, and the different brain structure is the cerebellar cortex, ependyma, or thalamus. A modified AAV capsid protein as described. wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 75, and said different brain structure is cerebellar cortex, ependyma, globus pallidus, optic nerve, substantia nigra, or 12. The modified AAV capsid protein of claim 1 or 11, which is the subthalamic nucleus. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 76, and the different brain structure is the cerebral cortex, ependyma, hippocampus, meninges, or substantia nigra. , the modified AAV capsid protein of claim 1 or 11. 12. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 77, and the different brain structure is the cerebral cortex, ependyma, or spinal cord. A modified AAV capsid protein as described. 12. The modified AAV capsid protein of claim 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 78, and the different brain structure is the cerebral cortex. AAV capsid protein. wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 79, and said different brain structure is cerebral cortex, hippocampus, optic nerve, or subthalamic nucleus. 12. The modified AAV capsid protein of paragraphs 1 or 11. 12. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 80, and the different brain structure is the ependyma, hippocampus, or meninges. A modified AAV capsid protein as described. wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is any one of SEQ ID NOs: 81-84 and 86, and said different brain structure is globus pallidus; 12. The modified AAV capsid protein of claims 1 or 11. 12. The claim 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 85, and the different brain structure is the globus pallidum or thalamus. A modified AAV capsid protein. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 87, and the different brain structure is the globus pallidum, optic nerve, substantia nigra, or subthalamic nucleus. , the modified AAV capsid protein of claim 1 or 11. said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 88, and said different brain structure is hippocampus, optic nerve, spinal cord, subthalamic nucleus, or thalamus; 12. The modified AAV capsid protein of claims 1 or 11. 12. The modified AAV of claim 1 or 11, wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 89, and said different brain structure is hippocampus. capsid protein. 1. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NOs: 90-93, and the different brain structure is the meninges. or the modified AAV capsid protein according to 11. 12. The modified AAV of claim 1 or 11, wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 94, and said different brain structure is the optic nerve. capsid protein. 12. The modification of claim 1 or 11, wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 95, and said different brain structure is putamen or spinal cord. AAV capsid protein that has been modified. 1. The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NOs: 96-100, and the different brain structure is the putamen. or the modified AAV capsid protein according to 11. 12. The modified AAV of claim 1 or 11, wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is SEQ ID NO: 101, and said different brain structure is the spinal cord. capsid protein. 12. Claims 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 102, and the different brain structure is the substantia nigra or subthalamic nucleus. modified AAV capsid proteins. 12. The modified AAV capsid protein of claim 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 103, and the different brain structure is the substantia nigra. AAV capsid protein. 12. The claim 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 104 or 105, and the different brain structure is the subthalamic nucleus. A modified AAV capsid protein. Claim 1 or wherein said modified AAV capsid protein is a modified AAV2 capsid protein, said targeting peptide is any one of SEQ ID NOs: 106-109, and said different brain structure is the thalamus; 11. A modified AAV capsid protein according to 11. wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 110, and said different brain structures are brain stem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, 16. The modified AAV capsid protein of claim 1 or 15, which is globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 111, and said different brain structures are brainstem, cerebellar cortex, cerebral cortex, ependyma, globus pallidum, 16. The modified AAV capsid protein of claim 1 or 15, which is hippocampus, meninges, optic nerve, substantia nigra, or subthalamic nucleus. wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 112, and said different brain structure is brainstem, globus pallidus, putamen, substantia nigra, or thalamus 16. The modified AAV capsid protein of claim 1 or 15, wherein the modified AAV capsid protein is wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 113, and said different brain structures are brain stem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, 16. The modified AAV capsid protein of claim 1 or 15, which is hippocampus, meninges, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 114, and the different brain structures are brainstem, cerebral cortex, globus pallidum, meninges, optic nerve, black 16. The modified AAV capsid protein of claim 1 or 15, which is cytoplasmic, or thalamic. 16. The claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 115, and the different brain structure is the brainstem or the caudate nucleus. A modified AAV capsid protein. The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 116, and the different brain structure is the brainstem, caudate nucleus, cerebral cortex, hippocampus, optic nerve, or brain structure. 16. The modified AAV capsid protein of claim 1 or 15, which is a shell. 16. The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 117, and the different brain structure is the brainstem, optic nerve, or substantia nigra. A modified AAV capsid protein as described. 2. The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 118, and the different brain structure is the caudate, ependyma, or meninges. or a modified AAV capsid protein according to 15. wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 119, and said different brain structures are caudate nucleus, cerebellar cortex, ependyma, globus pallidum, meninges , spinal cord, or subthalamic nucleus. wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 120, and said different brain structures are caudate, ependyma, globus pallidus, meninges, spinal cord, or subthalamic nucleus. 16. The modified AAV capsid protein of claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 121, and the different brain structure is the caudate nucleus. AAV capsid protein. wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 122, and the different brain structure is the cerebellar cortex, meninges, spinal cord, or subthalamic nucleus; 16. The modified AAV capsid protein of claims 1 or 15. 12. The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 123, and the different brain structure is the cerebellar cortex, hippocampus, putamen, or spinal cord. A modified AAV capsid protein according to 1 or 15. 16. The modified AAV capsid protein of claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 124, and the different brain structure is the cerebellar cortex. AAV capsid protein. 12. The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 125, and the different brain structure is the cerebellar cortex, cerebral cortex, hippocampus, or thalamus. A modified AAV capsid protein according to 1 or 15. 16. The modified AAV capsid protein of claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 126, and the different brain structure is the cerebral cortex. AAV capsid protein. 16. The claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 127, and the different brain structure is the cerebral cortex or putamen. A modified AAV capsid protein. 16. The modified AAV capsid protein of claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 128, and the different brain structure is the ependyma or spinal cord. AAV capsid protein. wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 129, and said different brain structure is globus pallidus, hippocampus, optic nerve, or substantia nigra. 16. The modified AAV capsid protein of paragraphs 1 or 15. 16. The modified AAV of claim 1 or 15, wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 130 and said different brain structure is hippocampus. capsid protein. 16. The modified AAV capsid protein of claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 131, and the different brain structure is the meninges. AAV capsid protein. 16. The claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 132, and the different brain structure is the optic nerve or subthalamic nucleus. A modified AAV capsid protein. 16. The modification of claim 1 or 15, wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 133, and said different brain structure is putamen or thalamus. AAV capsid protein that has been modified. 16. The modification of claim 1 or 15, wherein said modified AAV capsid protein is a modified AAV9 capsid protein, said targeting peptide is SEQ ID NO: 134, and said different brain structure is putamen or spinal cord. AAV capsid protein that has been modified. 16. The modified AAV capsid protein of claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 135, and the different brain structure is the subthalamic nucleus. AAV capsid protein. 16. The modified AAV capsid protein of claim 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 136 or 137, and the different brain structure is the thalamus. AAV capsid protein. A nucleic acid comprising a sequence encoding the modified capsid protein of any one of claims 1-157. A recombinant adeno-associated virus (rAAV) virus comprising the modified capsid protein of any one of claims 1-157. A viral vector comprising a nucleic acid encoding the modified capsid protein of any one of claims 1-157. 161. The viral vector of claim 160, further comprising a nucleic acid sequence encoding a nucleic acid of interest. 162. The viral vector of claim 161, wherein said nucleic acid of interest is a therapeutic agent. 163. The viral vector of claim 162, wherein said therapeutic agent is an enzyme or RNAi molecule. A cell comprising the viral vector of any one of claims 160-163. 165. The cell of claim 164, which is a mammalian cell. 165. The cell of claim 164, which is a human cell. 165. The cell of claim 164, which is in vitro. 165. The cell of claim 164, which is in vivo. 159. A pharmaceutical composition comprising the viral vector of claim 159 and a pharmaceutically acceptable carrier. 160. A method for delivering agents to different brain structures of a subject comprising administering the virus of claim 159 to the subject. A method for delivering an agent to the brain stem of a subject, the AAV1 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 1-9, SEQ ID NOs: 52-60. or an AAV9 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 110-117. described method. AAV1 Virus Comprising Modified Capsid Proteins with Targeting Peptides Selected from SEQ ID NOs: 1, 3, 7, and 10-16, SEQ ID NOs: 1, 3, 7, and 10-16 AAV2 viruses comprising modified capsid proteins with targeting peptides selected from ID NOs: 59 and 61-69, or targeting peptides selected from SEQ ID NOs: 110, 113, 115, 116, and 118-121 171. The method of claim 170, comprising administering an AAV9 virus comprising a modified capsid protein having a A method for delivering an agent to the cerebellar cortex of a subject, the AAV1 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 1, 3, 4, 9, and 17-21; AAV2 viruses comprising modified capsid proteins with targeting peptides selected from SEQ ID NOs: 56, 58, 60, and 70-75, or from SEQ ID NOs: 110, 111, 113, 119, and 122-125 171. The method of claim 170, comprising administering an AAV9 virus comprising modified capsid proteins with a selected targeting peptide. A method for delivering an agent to the cerebral cortex of a subject, the AAV1 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 1, 3, 5, 12, and 21-26; AAV2 viruses comprising modified capsid proteins with targeting peptides selected from SEQ ID NOs: 53, 58, 60, 62, 63, 66, and 76-79, or SEQ ID NOs: 110, 111, 113, 114 171. The method of claim 170, comprising administering an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from , 116, and 125-127. A method for delivering an agent to the ependyma of a subject comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 2-4, 7, 9, 21, 22, 27, and 28 AAV1 virus, AAV2 virus comprising modified capsid proteins with targeting peptides selected from SEQ ID NOs: 53, 60, 62, 63, 66, 74-77, and 80, or SEQ ID NOs: 110, 111, 171. The method of claim 170, comprising administering an AAV9 virus comprising modified capsid proteins with targeting peptides selected from 113, 118-120, and 128. A method for delivering an agent to the globus pallidum of a subject, wherein the modified agent has a targeting peptide selected from SEQ ID NOs: 3, 5, 12, 14, 16, 21, 22, and 29-31 an AAV1 virus comprising capsid proteins, an AAV2 virus comprising modified capsid proteins with targeting peptides selected from SEQ ID NOs: 60, 75, and 81-87, or SEQ ID NOs: 110-112, 114, 119, 171. The method of claim 170, comprising administering an AAV9 virus comprising a modified capsid protein with a targeting peptide selected from 120, and 129. A method for delivering an agent to the hippocampus of a subject, the AAV1 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 1-4, 7, and 32-34, and 28; An AAV2 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 53, 55, 58, 60, 63, 76, 79, 80, 88, and 89, or SEQ ID NOs: 110, 111 171. The method of claim 170, comprising administering an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from , 113, 116, 123, 125, 129, and 130. A method for delivering an agent to the meninges of a subject, the modified having a targeting peptide selected from SEQ ID NOs: 3, 5, 7, 9, 12, 21, and 35-37, and 28 AAV1 virus comprising capsid proteins, AAV2 viruses comprising modified capsid proteins with targeting peptides selected from SEQ ID NOs: 53, 58, 60, 66, 73, 76, 80, and 90-93, or SEQ ID NOs: 171. The method of claim 170, comprising administering an AAV9 virus comprising a modified capsid protein with a targeting peptide selected from NO: 110, 111, 113, 114, 118, 119, 122, and 131. A method for delivering an agent to the optic nerve of a subject, comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 2, 3, 7, 14-16, 21, 31, and 38 AAV1 virus, AAV2 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NO: 53, 54, 57, 58, 60, 75, 79, 87, 88, and 94, or SEQ ID NO: 171. The method of claim 170, comprising administering an AAV9 virus comprising modified capsid proteins with targeting peptides selected from 110, 111, 114, 115, 117, 129, and 132. A method for delivering an agent to the putamen of a subject comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 3, 4, 12, 13, 21, 30, and 39-42. an AAV1 virus comprising, an AAV2 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 55, 59, 60, 61, and 95-100, or SEQ ID NOs: 110, 112, 113, 116 171. The method of claim 170, comprising administering an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from , 123, 127, 133, and 134. A method for delivering an agent to the spinal cord of a subject, comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 2-4, 7, 9, 21, 32, 33, and 43 AAV1 virus, AAV2 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 53, 58-61, 63, 77, 88, 95, and 101, or SEQ ID NOs: 110, 113, 171. The method of claim 170, comprising administering an AAV9 virus comprising modified capsid proteins with targeting peptides selected from 119, 120, 122, 123, 128, and 134. A method for delivering an agent to the substantia nigra of a subject, the AAV1 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 2, 3, 9, 44, and 45, and 28 , an AAV2 virus comprising a modified capsid protein with a targeting peptide selected from SEQ ID NOs: 52, 53, 57, 58, 75, 76, 87, 102, and 103, or SEQ ID NOs: 110-114, 171. The method of claim 170, comprising administering an AAV9 virus comprising a modified capsid protein with a targeting peptide selected from 117, and 129. A method for delivering an agent to the subthalamic nucleus of a subject, comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 2-4, 12, 16, 30, 46, and 47 AAV1 virus, SEQ ID NO: 57, 58, 60, 75, 79, 87, 88, 102, 104, and 105 AAV2 virus comprising a modified capsid protein with a targeting peptide selected from, or SEQ ID NO: 171. The method of claim 170, comprising administering an AAV9 virus comprising modified capsid proteins with targeting peptides selected from 110, 111, 113, 119, 120, 122, 132, and 135. A method for delivering an agent to the thalamus of a subject comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 1, 2, 8, 12, 21, 28, and 48-51 AAV1 virus, AAV2 virus comprising modified capsid proteins with targeting peptides selected from SEQ ID NOs: 52, 55, 56, 74, 85, 88, and 106-109, or SEQ ID NOs: 110, 112- 171. The method of claim 170, comprising administering an AAV9 virus comprising modified capsid proteins with targeting peptides selected from 114, 125, 133, 136, and 137. 185. The method of any one of claims 170-184, wherein said agent is an siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system. 185. The method of any one of claims 170-184, wherein said administering is to the central nervous system. 187. The method of claim 186, wherein said administration is to the cisterna magna, intraventricular space, ependymal, ventricle, subarachnoid space, cochlea, and/or intrathecal space. said ventricle is the rostral ventricle and/or the caudal ventricle and/or the right ventricle and/or the left ventricle and/or the right rostral ventricle and/or the left rostral ventricle 188. The method of claim 187, wherein the lateral ventricle and/or the right caudal ventricle and/or the left caudal ventricle. 189. The method of any one of claims 170-188, wherein a plurality of viral particles are administered. 189. The method of claim 189, wherein said virus is administered at a dose of about 1 x ¹⁰⁶ to about 1 x 1018 vector genomes per kilogram ⁽ vg/kg). The virus is about 1×10 ⁷ to 1×10 ¹⁷ , about 1×10 ⁸ to 1×10 ¹⁶ , about 1×10 ⁹ to 1×10 ¹⁵ , about 1×10 ¹⁰ to 1×10 vg per kg of patient. ¹⁴ , about 1×10 ¹⁰ to 1×10 ¹³ , about 1×10 ¹⁰ to 1×10 ¹³ , about 1×10 ¹⁰ to 1×10 ¹¹ , about 1×10 ¹¹ to 1×10 ¹² , about 1×10 189. The method of claim 189, administered at a dose of ¹² to x ¹⁰¹³ , or about ¹ x ¹⁰¹³ to 1 x 1014. 192. The method of any one of claims 170-191, wherein said subject is a human. 159. A method of treating disease in a mammal comprising administering the virus of claim 159 to said mammal. 194. The method of claim 193, wherein said disease is a neurodegenerative disease. 194. wherein said neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, Alzheimer's disease, polyglutamine repeat disease, or Parkinson's disease described method. 194. The method of claim 193, wherein said mammal is human.

Images